hUNG2 Is the Major Repair Enzyme for Removal of Uracil from
U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with
hSMUG1 as a Broad Specificity Backup*
Bodil
Kavli,
Ottar
Sundheim,
Mansour
Akbari,
Marit
Otterlei,
Hilde
Nilsen
,
Frank
Skorpen,
Per Arne
Aas,
Lars
Hagen,
Hans E.
Krokan, and
Geir
Slupphaug§
From the Institute of Cancer Research and Molecular Biology,
Norwegian University of Science and Technology,
N-7489 Trondheim, Norway
Received for publication, July 16, 2002
 |
ABSTRACT |
hUNG2 and hSMUG1 are the only known glycosylases
that may remove uracil from both double- and single-stranded DNA in
nuclear chromatin, but their relative contribution to base excision
repair remains elusive. The present study demonstrates that both
enzymes are strongly stimulated by physiological concentrations of
Mg2+, at which the activity of hUNG2 is
2-3 orders of magnitude higher than of hSMUG1. Moreover,
Mg2+ increases the preference of hUNG2 toward uracil in
ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1
against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than
hUNG2, including 5-hydroxymethyluracil and
3,N4-ethenocytosine. hUNG2 is excluded from,
whereas hSMUG1 accumulates in, nucleoli in living cells. In
contrast, only hUNG2 accumulates in replication foci in the S-phase.
hUNG2 in nuclear extracts initiates base excision repair of plasmids
containing either U:A and U:G in vitro. Moreover, an
additional but delayed repair of the U:G plasmid is observed that is
not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We
propose a model in which hUNG2 is responsible for both prereplicative
removal of deaminated cytosine and postreplicative removal of
misincorporated uracil at the replication fork. We also provide
evidence that hUNG2 is the major enzyme for removal of deaminated
cytosine outside of replication foci, with hSMUG1 acting as a
broad specificity backup.
| |
INTRODUCTION |
Uracil in DNA can be introduced via two mechanisms, deamination of
cytosine and misincorporation of dUMP during replication. Deamination
of cytosine has been calculated from measured deamination rates to
occur at a rate of 100-500 per human cell/day (1, 2) to yield
mutagenic U:G mispairs. Uracil may also appear as a consequence of
misincorporation of dUMP instead of dTMP during replication, resulting
in a U:A base pair. The latter is not miscoding, but may produce
cytotoxic and mutagenic AP site intermediates during repair. In
organisms containing 5-methylcytosine in their genomes, deamination of
5-methylcytosine furthermore leads to T:G mismatches. All living
organisms express uracil-DNA glycosylases (UDGs)1 that prevent
cytotoxic and mutagenic effects of the above lesions. UDGs remove
uracil (and sometimes other damaged bases or thymine) from the
deoxyribose and thus initiate a multistep base excision repair (BER)
pathway, eventually restoring the correct DNA sequence. After removal
of uracil by an UDG and cleavage of the resulting abasic site by AP
endonuclease (APE1/APE2), the BER pathway splits into two branches
(reviewed in Ref. 3). The presumed major track is the short-patch
pathway. It uses the 5'- deoxyribophosphodiesterase activity of
DNA polymerase 
to cleave 3' of the abasic site, thus releasing
deoxyribose-5-phosphate. Then pol inserts C or T, depending on the
template base. Finally, DNA ligase III seals the nick, perhaps aided by
the scaffold protein XRCC1. The alternative long-patch pathway largely
uses replication proteins and may take place in replication foci (4).
This pathway requires pol 
and/or 
, as well as the trimeric
sliding clamp and polymerase processivity factor proliferating cell
nuclear antigen (PCNA) and the clamp loader replication factor C (RFC).
Repair synthesis is stimulated by pol , which may be important in
the first step of polymerization. The structure-specific endonuclease
FEN1 removes the 2-8-nucleotide displaced "flap" of DNA, and DNA
ligase I seals the nick (3).
Mammalian cells contain at least four UDGs, of which three (UNG, SMUG1,
and TDG) belong to the same protein superfamily, possess the same fold,
and have probably evolved from a common ancestor (5). Of these, UNG
appears to be quantitatively dominating as determined from activity
assays using human cell-free extracts and U:A substrates (6). UNG
belongs to the family of highly conserved UDGs typified by
Escherichia coli Ung, and is present in a large number of
eukaryotes, bacteria, and large eukaryotic DNA viruses (7). The human
and mouse UNG genes encode both mitochondrial (UNG1) and
nuclear (UNG2) forms of the enzyme by way of alternative promoter usage
and mRNA splicing (8). The catalytic domain of hUNG has been
extensively studied, and its structure and molecular mechanism of
catalysis and specificity established (9-12). The enzyme removes
uracil in vitro in the order of preference ssU > U:G > U:A (6). Certain closely related bases formed from cytosine
after 
-irradiation or oxidative stress are also substrates, such as
5-hydroxyuracil, isodialuric acid, and alloxan (13). These are,
however, excised at a very low rate compared with uracil. A second
human UDG against both ss- and dsU, hSMUG1 (single-strand selective
monofunctional uracil-DNA glycosylase), was identified more recently by
in vitro expression cloning (14). SMUG1 is not found in
bacteria and yeast, but is present in higher eukaryotes. Although they
probably share the same fold and motifs necessary for substrate binding
and catalysis as hUNG, little homology exists between the two enzymes
at the amino acid level (5). hSMUG1 is furthermore located in the nuclei, and Xenopus SMUG1 has a substrate preference similar
to the catalytic hUNG domain (ssU > U:G > U:A), although
its specific activity is considerably lower (14). Recently, Boorstein
and co-workers (15) demonstrated that hSMUG1 is also active against 5-hydroxymethyluracil (HmU:A). HmU is formed in DNA by oxidative attack
on the methyl group of thymine, thereby creating HmU:A. It is also the
product of the deamination of 5-hydroxymethylcytosine, which may be
formed via oxidation of 5-methylcytosine. The latter creates a HmU:G
base pair, which would be mutagenic if left unrepaired. Although HmU:G
substrates were not tested, the authors suggested the latter could be a
biologically important substrate (15). This is also supported by the
fact that transition mutation from 5mC:G to T:A is the most
frequent substitution mutation in human cancer (16). The two last UDGs
identified in human cells, TDG and MBD4 (MED1), are both
mismatch-specific and have no activity against single-stranded
substrates. TDG excises uracil and thymine from U:G and T:G mismatches,
as well as 3,N4-ethenocytosine (C) and
5-fluorouracil (5-FU) from double-stranded DNA, and may restore G:C
base pairs at sites of cytosine or 5-methylcytosine deamination, or
alkylation, respectively (17). MBD4, which does not belong to the same
superfamily as the three other UDGs, acts on uracil, thymine, 5-FU, and
C mispaired with guanine (18), as well as on 5-methylcytosine at
hemimethylated DNA (19). The preferred substrates, however, are G:T
mismatches at methylated or unmethylated CpG islands. Thus, MBD4, as
well as TDG, may have a function in the correction of T:G mismatches
originating from deamination of 5-methylcytosine.
Several lines of evidence indicate that nuclear UNG2 has a major role
in postreplicative removal of misincorporated uracil in mammalian cells
(4). The contribution of UNG2 to repair of deaminated cytosines has,
however, been debated. Whereas bacterial and yeast
ung
mutants display a mutator phenotype unable
to repair deaminated cytosines (20, 21), such a phenotype is not
clearly observed in Ung/ mice (22). Based on
this finding, and comparison of kinetic parameters of the
Xenopus SMUG1 (14) and the hUNG catalytic domain (6), it was
suggested that in higher eukaryotes, the contribution of UNG2 to the
excision of deaminated cytosines was reduced, and that this function
might instead be provided by SMUG1 (23). In human cells, however, the
UNG catalytic domain alone is not observed in the nucleus (24). Rather,
the entire N-terminal regulatory domain remains attached to the core
catalytic domain after nuclear translocation. Until now, little
information has existed on the enzymatic properties of full-length
hUNG2. A likely reason for this is the susceptibility of the enzyme to
N-terminal proteolytic degradation during purification, mainly
resulting in the core catalytic domain (25). To gain further insight in the functional properties of hUNG2 and hSMUG1 and their relative contribution to nuclear base excision repair, both proteins were purified after overexpression in E. coli. In depth
biochemical characterization and analysis of subnuclear localization
revealed previously unrecognized properties of both hUNG2 and hSMUG1
that may have important implications for their functions in nuclear BER in vivo.
| |
EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant hUNG2--
An
NdeI site was introduced at the hUNG2 translation
start codon, and a 947-bp NdeI/HpaI fragment
encompassing the entire hUNG2 coding region was ligated into
the new vector pJB658cop251kan. This expression vector was
constructed by introducing a high copy number variant of the
trfA gene (Arg-251 
Met) and changing the resistance from
ampicillin to kanamycin in the toluic acid-inducible, broad host vector
pJB658 (26). The resultant construct, p658KanUng2 was introduced into
E. coli BL21. Fermentation, preparation of crude extract,
and the initial chromatographic steps were essentially as described for
the hUNG catalytic domain (6) except that the culture was induced by 1 mM toluic acid (final) and allowed to grow for additionally
3 h at 30 °C before harvesting. Furthermore, Complete® mini (EDTA-free) (Roche) protease inhibitor
tablets were included in the initial steps of the purification (1 tablet/10 ml during homogenization and 1 tablet/l ml in subsequent
buffers). During size exclusion chromatography on Superdex 75 26/60
(Amersham Biosciences), buffer was changed to 20 mM
HEPES-NaOH (pH 8.0), 100 mM NaCl, 1 mM DTT.
Pooled active fractions were loaded onto a MonoS HR 5/5 column, and
after washing to base-line absorbance, bound proteins were eluted using
a linear NaCl gradient from 100 mM to 1 M NaCl in the same buffer. To remove partially N-terminally deleted species, the purest fractions were subjected to MonoS rechromatography and
fractions containing the highest portion of apparently full-length protein were pooled. The purified protein was finally concentrated by
ultrafiltration and snap-frozen in liquid N2 prior to
storage at 
80 °C.
Expression and Purification of Recombinant hSMUG--
IMAGE
clone identification no. 726197 containing the hSMUG1 cDNA in
pT7T3D was cut using NdeI and BfaI, and the
1154-bp fragment encoding hSMUG1 was cloned into the NdeI
site of pET11a (Invitrogen). The resultant vector was transformed into
E. coli BL21-CodonPlus(DE3)-RIL (Stratagene) and bacterial
cell mass for purification produced by fermentation. Expression was
induced by 1 mM
isopropyl-1-thio--D-galactopyranoside (final) at
37 °C and the culture allowed to grow for additional 4 h prior
to cell harvest. Preparation of the hSMUG1 crude extract was
essentially as described for the hUNG catalytic domain (6), except that
homogenization and protamine sulfate precipitation was performed in 20 mM HEPES-NaOH (pH 6.8), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 1 tablet/10 ml
Complete® mini (EDTA-free) protease inhibitor mixture
(buffer A). The protamine sulfate fraction was loaded onto a
DEAE-Sephacel column (Amersham Biosciences, 5 × 9 cm) coupled in
series with a CM-Sepharose column (Amersham Biosciences, 5 × 8.5 cm). After washing to base-line absorbance, the DEAE-Sephacel column
was bypassed and adsorbed proteins were eluted in a 0.05-1
M NaCl gradient in buffer A. Active fractions were pooled
and dialyzed against 20 mM HEPES-NaOH (pH 8), 10 mM NaCl, 1 mM DTT (buffer B) and applied to a
UnoS-12 column (Bio-Rad). Adsorbed proteins were eluted in a 0.01-0.7 M linear NaCl gradient. Active fractions were pooled and
concentrated by ultrafiltration to 5 ml and loaded onto a Superdex 75 HiLoad 26/60 column (Amersham Biosciences) preequilibrated with buffer B containing 100 mM NaCl, and eluted with the same buffer.
Fractions containing hSMUG1 were verified by SDS-PAGE and pooled. After a 4-fold dilution in buffer B, hSMUG1 was loaded onto a
poly(U)-Sepharose column (Amersham Biosciences, 1.6 × 10 cm)
preequilibrated with buffer B containing 2 mM EDTA.
Adsorbed proteins were eluted in a 0.01-1 M linear NaCl
gradient in the same buffer and the fractions containing hSMUG1
identified by SDS-PAGE and pooled. The poly(U)-Sepharose fraction was
then dialyzed against buffer B, applied onto a MonoS HR 5/5 column, and
eluted in a linear 0.01-1 M NaCl gradient in the same
buffer. The purest hSMUG1 fractions were collected, and the remaining
hSMUG1-containing fractions rechromatographed on MonoS as above. The
pooled hSMUG1 fraction was apparently homogeneous as determined by
SDS-PAGE and silver staining. The purified protein was finally
concentrated by ultrafiltration and snap-frozen in liquid
N2 prior to storage at 80 °C.
Neutralizing Antibodies against hUNG2 and hSMUG1--
Polyclonal
PU101 against the hUNG catalytic domain was prepared as described (6).
Polyclonal PSM1 against hSMUG1 was prepared by subcutaneous injection
of 66 µg of purified, recombinant hSMUG1 in Freund's complete
adjuvant at the neck and flank of New Zealand White rabbits. Subsequent
immunizations with the same amount of hSMUG1 in incomplete Freund's
adjuvant were performed as above at 3-week intervals, and the final
bleed 10 days after the last immunization. IgG-fractions of PU101 and
PSM1 were purified on protein A-Sepharose HiTrap columns (Amersham
Biosciences) before being used in inhibition experiments.
Cell Culture and Preparation of Nuclear
Extracts--
Spontaneously transformed human keratinocytes HaCaT,
colorectal carcinoma CX-1, and HeLa cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 0.03% glutamine, and 0.1 mg/ml gentamicin at 5% CO2. Cells were harvested
in the logarithmic growth phase by trypsinization, washed twice in
ice-cold phosphate-buffered saline prior to isolation of nuclei.
Subsequently, CX-1 and HeLa were washed once in ice-cold isotonic
buffer (20 mM HEPES-NaOH (pH 7.8), 1 mM
MgCl2, 5 mM KCl, 1 mM DTT, and 250 mM sucrose). The pellet was resuspended in hypotonic buffer
(isotonic buffer without sucrose) containing Complete®
protease inhibitor mix (1 tablet/50 ml) and left on ice for 45 min to
swell. Cells were lysed by 10-20 strokes in a Dounce homogenizer (tight-fitting pestle), and nuclei pelleted by centrifugation at
600 × g for 6 min. The pellets were resuspended in
hypertonic buffer (hypotonic buffer with 0.5 M NaCl and
25% glycerol), and incubated at 4 °C on a rotary shaker at high
speed for 30 min to extract nuclear proteins. Nuclear debris was
removed by centrifugation at 12,000 × g for 15 min,
protein concentration in the supernatant measured using the Bio-Rad
protein assay (BSA as standard), and the nuclear extract aliquoted,
snap-frozen in liquid N2, and stored at 80 °C until
use. Nuclei from HaCaT keratinocytes were prepared using the
NuClear® nuclear extraction kit (Sigma) according to the
protocol form the manufacturer, using isotonic buffer and detergent.
Serum starvation, [3H]thymidine pulse labeling, and
harvesting of HaCaT cells were as described (27). Polyclonal PSM1
antiserum was covalently coupled to magnetic protein A Dynabeads
(Dynal, Norway) according to the instructions from the manufacturer.
Cells (5 × 105) from each time point were resuspended
in 1.5 ml of lysis buffer (1× Tris-buffered saline, 1 mg/ml BSA, 1%
Triton X-100, 1 tablet/50 ml Complete® maxi+EDTA (Roche)
protease inhibitors) and passed 10 times through a 21-gauge syringe
needle. PSM1-coupled magnetic beads (10 µl) were added, and the
extract incubated on a rotary shaker for 2 h at 4 °C. After
extensive washing, the beads were eluted by boiling in SDS loading
buffer and subjected to SDS-PAGE. After electroblotting to
polyvinylidene difluoride membranes, bands were visualized by standard
Western analysis using PSM1 as primary antibodies, HRP swine × rabbit secondary antibodies (Dako), and ECL (Amersham Biosciences).
Cloning and Expression of ECFP and EYFP Constructs--
PCR was
run on DNA from IMAGE clone cDNA_id 726197 encoding hSMUG1 in
pT7T3D, using Pfu polymerase (Stratagene), a T7 primer, and
the primer 5'-ATT TCA ACA GCA GTG GCA GC-3'. The PCR product was
purified by QIAQuick PCR purification (Qiagen) and digested with
EcoRI. The PCR fragment (EcoRI/blunt) encoding
hSMUG1 was cloned into EcoRI/SmaI of pEGFP-N1
(Clontech). hSMUG1 and hUNG2 cDNA from pSMUG1-EGFP and pUNG2EGFP
(4) were ligated into pEYFP-N1 (Clontech) by transferring the
NheI/AgeI fragment into the corresponding sites
in pEYFP-N1 to make SMUG1-EYFP and UNG2-EYFP, respectively. To make the
C-terminal fusion of SMUG1 with EYFP, the
EcoRI/BamHI fragment from SMUG1-EYFP was ligated
with EcoRI/BamHI-digested pEYFP-C1 (Clontech).
The resulting vector was digested with SacI, the 3' overhang
removed by T4 DNA polymerase, and the vector re-ligated to make
EYFP-SMUG1. The fusion constructs were verified to be in-frame by sequencing.
The construct carrying the hUNG2 promoter, pGL2-PA (27),
was digested with HindIII/EcoRV to remove the
luciferase gene. The HindIII/DraI fragment from
pUNG2-EYFP was then ligated with HindIII/EcoRV-digested pGL2-PA. From
this plasmid, the SacI/NotI fragment containing
ProA-UNG2-EYFP was excised, and ligated into SacI/NotI-digested pEGFP-1 (Clontech), thus
replacing the EGFP-coding sequence in the vector and giving
pProA-UNG2-EYFP. ECFP-PCNA fusion constructs were prepared from
pEGFP-PCNAL2 (28), which contains the SV40 NLS in front of PCNA in a
C-terminal fusion with EGFP. The SV40 NLS was amplified by using PCR
primers containing the sites for the restriction enzymes
NheI and AgeI. After digestion with
NheI and AgeI, the fragment was cloned into the
corresponding sites in the pECFP-C1 vectors (Clontech). Next, the
BsrGI (in EGFP)/XbaI (behind PCNA) fragment from
the pEGFP-PCNAL2 construct was cloned into the
BrsGI/XbaI sites of pECFP-C1 vector with the SV40
NLS N-terminally, giving pNLS-ECFP-PCNA (named pECFP-PCNA). HeLa cells
were transiently and stably transfected by using calcium phosphate
(Profection, Promega) according to the recommendations from the
manufacturer. Stable transfectants of HeLa with pProA-UNG2-EYFP were
further cloned by use of limited dilutions in 96-microwell dishes in
medium containing G418. The cells were examined in a Zeiss LSM 510 laser-scanning microscope equipped with a Plan-Apochromat 63×/1.4 oil
immersion objective. The 458-nm laser line was used for excitation of
ECFP (detected at 480 nm < 
ECFP < 520 nm) and the
514-nm laser line for EYFP (detected at EYFP > 560 nm).
UDG Activity Assays--
Unless otherwise stated, UDG activity
was measured in 20 µl of assay mixture containing (final) 20 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mg/ml bovine serum
albumin, 1.8 µM [3H]dUMP-containing calf
thymus DNA (specific activity 0.5 mCi/µmol) and varying amounts of
enzyme. The mixture was incubated 10 min at 30 °C, and the amount of
released uracil measured as described (29). Kinetic assays using
0.24-21.5 µM calf thymus substrate were performed in the
buffer above. In separate experiments kinetic parameters were measured
in the presence of either 7.5 mM MgCl2 or 7.5 mM MgCl2 and 0.44 ng/µl APE1 in the same
buffer (molar ratio of APE1/glycosylase at least 10/1). The amount of
hUNG2 or hSMUG1 used in the kinetic assays was adjusted to ensure that less than 30% of the substrate was consumed, to ensure linearity of
the assay. Kinetic parameters were calculated using the Enzpack for
Windows version 1.4 software package (Biosoft) using the method of
Wilkinson. Enzyme kinetic parameters were also analyzed using a panel
of oligonucleotide substrates. These were prepared by 33P-5'-end labeling of 19-mer PAGE-purified
oligonucleotides containing U or HmU at a central position
(U141, 5'-CATAAAGTGUAAAGCCTGG-3'; HmU141, 5'-CATAAAGTGHmUAAAGCCTGG-3').
To generate double-stranded substrates, the labeled strands were
annealed to a 50% excess of the complementary strand containing either
A or G opposite U or HmU. Varying substrate concentrations (0.1-15
µM) were made by addition of identical non-labeled
oligonucleotides. The kinetic assays (10 µl total) were performed in
the same buffer as for the [3H]dUMP-containing calf
thymus DNA in the presence of MgCl2. After 10 min of
incubation at 30 °C, the reactions were stopped by addition of 50 µl of 1 M piperidine and heated at 90 °C for 20 min to
cleave at AP sites. Piperidine was removed by drying under vacuum, and the oligonucleotides redissolved in 65% formamide loading buffer and
analyzed by denaturing PAGE (12% polyacrylamide, 7 M
urea). Cleaved and uncleaved oligonucleotides were quantified by
phosphorimaging. Kinetic parameters were calculated as above. In
separate experiments, the activities of hUNG2 and hSMUG1 were directly
compared by using ds oligonucleotides (20 nM final) having
the same sequence as above, and containing U:G, HmU:G, 5-fluorouracil:A
(FU:A), 3,N4-ethenocytosine:G (C:G), or
5-hydroxyuracil:G (5-OHU:G). To detect activity against the weaker
substrates, reactions contained varying amounts of hUNG2 or hSMUG1
(0-10 ng/µl final) and were incubated at 37 °C for 30 in the
presence of 7.5 mM Mg2+ with or without APE1
(0.1 ng/µl final). Inhibition of hSMUG1 and hUNG2 by polyclonal
antibodies or oligonucleotides containing AP sites was monitored after
incubation of the purified enzymes or cell extracts with inhibitor for
10 min on ice prior to the enzyme assays.
BER Assay--
Double-stranded plasmid DNA substrates containing
uracil at defined positions were prepared essentially as described
(30). Briefly 20 µg of ssDNA (pGEM-3zf+) was annealed to 4.2 µg of
a 22-mer complementary oligonucleotide containing uracil and synthesis of duplex DNA carried out in the presence of T4 DNA polymerase, T4 DNA
ligase, and T4 gene 32 ssDNA-binding protein at 37 °C for 2h. Closed
circular DNA duplex molecules were purified by CsCl gradient centrifugation.
The base excision repair mixtures (50 µl) contained (final) 40 mM HEPES-KOH (pH 7.8), 70 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 2 mM ATP, 20 µM dATP, 20 µM dGTP,
8 µM dTTP or dCTP depending on the isotope used, 8 µM phosphocreatine, 0.36 mg/ml BSA, 1 µg/ml creatine
phosphokinase, 40 µCi/ml [
-32P]dTTP or
[-32P]dCTP, 0.6 or 0.2 mg/ml total cell or nuclear
extract, respectively (measured as protein), and 6 µg/ml dsDNA
template. The repair mixtures were incubated at 30 °C for the given
times, stopped by adding EDTA and RNase A, and further incubated with
proteinase K (30 min) and SDS (10 min) at 37 °C. The repair product
was recovered by phenol/chloroform extraction and ethanol/salt
precipitation. The DNA was digested using
XbaI/HincII for short-patch and
HincII/PstI for long-patch BER, and repair
products were analyzed by electrophoresis in 12% denaturing
polyacrylamide gels and phosphorimaging of the dried gels.
To specifically monitor the glycosylase and AP endonuclease steps in
the BER-assays, 50-mer double stranded oligonucleotides were prepared
that corresponded to the region of the BER plasmid substrates
containing the U:A or U:G lesions (uracil-containing strand:
5'-TCGGTACCCGGGGATCCTCTAGAGXYGACCTGCAGGCATGCAAGCTTGAG-3', with X representing uracil in the U:A oligo, and
Y representing uracil in the U:G oligo). The duplexes were
incubated in the presence of the same amounts of nuclear extract and in
the same buffer as in the BER assay, except that dNTPs were replaced by
ddCTP or ddTTP to arrest polymerization and ligation. The amount of AP
sites or incised product was quantified as described above.
| |
RESULTS |
Purification of Recombinant hUNG2 and hSMUG1--
We have found
full-length hUNG2 to be notoriously difficult to express and purify
from various E. coli strains and budding yeast
(Pichia) caused by N-terminal proteolysis.
Apparently, this was caused by both in vivo proteolytic
attack and degradation during purification (data not shown). The
degradation problem was partially overcome by expressing the protein
containing an N-terminal His tag. For functional analysis we decided to
avoid histidine tags, however, because this may seriously affect the functional properties of some proteins (31). In the present system, the
highest relative amount of full-length hUNG2 at the time of cell
harvest was observed using E. coli BL21 (ompT
, lon), in which ~50% of the
protein appeared unprocessed. The presence of mixed protease-inhibitors
during purification only partially prevented further loss of the intact
protein, which generated enzyme species lacking one or a few of the
N-terminal residues. These results indicate that the hUNG2 N-terminal
regulatory region constitutes a distinct domain sensitive to
proteolysis. This would also explain why previous attempts to purify
UNG from mammalian cells in the absence of appropriate protease
inhibitors have yielded essentially the catalytic domain (25). This is
also corroborated by the abnormal chromatographic behavior of hUNG2.
The full-length protein (34.6 kDa) eluted as a 52-kDa protein in
size-exclusion chromatography, whereas the compact, spherical catalytic
domain (25.5 kDa) eluted as a 20-kDa protein (data not shown).
Furthermore, hUNG2 eluted over a broad NaCl range in MonoS
chromatography, in contrast to the sharp peak observed with the
catalytic domain alone. This may indicate that the hUNG2 N-terminal is
less ordered than the catalytic domain, and may adopt alternative conformations.
The purified enzyme after repeated MonoS chromatography was apparently
homogeneous as judged by SDS chromatography and silver staining.
N-terminal sequencing, however, revealed that this fraction contained
~60% full-length hUNG2, and ~40% of UNG2
1 lacking the N-terminal methionine. This was judged adequate for biochemical characterization of the enzyme, and no further attempts were made to
remove the 1-amino acid truncated form. Approximately 1 mg of purified,
full-length hUNG2 was obtained from a 5-liter fermentation. hSMUG1 was
expressed as a full-length protein. However, the hSMUG1 N terminus also
proved to be highly susceptible to proteolytic attack even in the
presence of mixed protease inhibitors, and virtually all hSMUG1
obtained after purification proved to lack the 16 N-terminal amino
acids. The yield was ~5 mg of protein/5 liters of fermenter culture.
No significant difference in enzymatic activity was observed with the
16 form as compared with full-length hSMUG1 expressed using another
vector system (pTYB12; data not shown). The former was, however, chosen
for biochemical analysis because of the very low yield from the pTYB12 system.
Both hUNG2 and hSMUG1 Are Stimulated by Mg2+ and
APE1--
To analyze the kinetic properties of hUNG2 and
hSMUG1, we first established reaction conditions under which
both activities could be directly compared. The pH profile of each
enzyme was broad, with an optimum between pH 7.0 and 7.5 (data not
shown). Furthermore, hUNG2 was stimulated ~2-fold by 50 mM NaCl (Fig. 1A),
whereas hSMUG1 was ~30% stimulated at 70 mM NaCl (Fig.
1B). Both the pH profiles and NaCl optima are similar to
those reported for the major UDG activity partially purified from HeLa
cells (29). In the latter study, the authors also observed a weak stimulation of the activity by Mg2+. To our knowledge, this
has not been reported for any other monofunctional DNA glycosylase,
including UNG, and it was thus of interest to analyze whether this
could be caused by stimulation of co-purifying hSMUG1 in their study.
To our surprise, however, both enzymes were markedly stimulated by the
presence of Mg2+. In the absence of monovalent cations,
hUNG2 was stimulated nearly 10-fold in the presence of 10 mM MgCl2 (Fig. 1A), whereas a near 2-fold stimulation was observed for hSMUG1 (Fig. 1B). This
is in contrast to properties of the catalytic core form of hUNG, which
is inhibited by Mg2+ at all concentrations tested (data not
shown). Thus the N-terminal 86 amino acids in hUNG2 are required for
the observed Mg2+-stimulation. Because hSMUG1 and hUNG2 are
both nuclear proteins, and the hUNG2 N-terminal region is retained
after nuclear translocation (24, 32), we decided to undertake an
in-depth study of the kinetic effects of magnesium on both enzymes. An
additional advantage of studies of effects of Mg2+ on hUNG2
and hSMUG1 was that their properties could be analyzed in the presence
of fully active APE1, which catalyzes the next step in the BER pathway.
APE1 has been demonstrated to affect the catalytic rate of several DNA
glycosylases (33, 34) and requires Mg2+ for full catalytic
activity, but not for DNA binding (35).
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Fig. 1.
Modulation of hUNG2 and hSMUG1 activities by
NaCl, Mg2+, and APE1. The activities of hUNG2 and
hSMUG1 against [3H]dUMP-containing calf thymus DNA were
analyzed in the presence of varying concentrations of NaCl or
MgCl2 (A and B), or with varying
concentrations of MgCl2 in the presence or absence of APE1
(C and D). A, hUNG2 activity with
dsDNA in the presence of varying concentrations of NaCl
(filled circles) and MgCl2
(open circles). B, hSMUG1 activity
with dsDNA in the presence of varying concentrations of NaCl
(filled circles) or MgCl2
(open circles). C, hUNG2 activity
against ssDNA (triangles) and dsDNA (rectangles) in the
presence (filled symbols) or absence
(open symbols) of APE1. D, hSMUG1
activity against ssDNA (triangles) and dsDNA
(rectangles) in the presence (filled
symbols) or absence (open symbols) of
APE1.
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The effects of Mg2+ on single-stranded (ssU) and
double-stranded (dsU) DNA substrates in the absence and presence of
APE1 are shown in Fig. 1, C (hUNG2) and D
(hSMUG1). In the absence of Mg2+, the activities of hUNG2
against ss- and ds- [3H]dUMP-containing calf thymus DNA
(U opposite A) were essentially identical. Moreover, a weak stimulation
of the activity against dsU was observed in the presence of APE1.
Increasing the Mg2+ concentration resulted in a gradual
increase in activity up to 6 and 10 mM Mg2+ for
dsU and ssU, respectively. In addition, the stimulatory effect by APE1
markedly increased for dsU in the presence of Mg2+ (Fig.
1C). A somewhat different pattern was observed for hSMUG1 (Fig. 1D). In the absence of Mg2+, hSMUG1 had
~4-fold higher activity against ssU than dsU. The activity against
both substrates increased with increasing Mg2+ up to 6 mM, although the relative ssU/dsU activity did not change. As for hUNG2, the presence of APE1 had only a weak stimulatory effect
with both substrates in the absence of Mg2+. However, in
the presence of Mg2+, APE1 had a marked stimulatory effect.
This was most pronounced for dsU, as the activity was stimulated
~10-fold by APE1 in the presence of 10 mM
Mg2+.
In separate experiments, potential stimulation of hUNG2 and hSMUG1 by
the second human AP endonuclease APE2 and the Flap endonuclease FEN1
was investigated. Neither of these proteins, however, had any effect on
the glycosylase activities (data not shown).
Mg2+ Strongly Increases the Affinity of hUNG2 for
Uracil in Single-stranded DNA--
To further analyze this complex
pattern of stimulation, kinetic assays were performed both in the
presence and absence of Mg2+ and APE1. Based on the above,
the reaction conditions were kept identical for hSMUG1 and hUNG2 (see
"Experimental Procedures"). The results are shown in Table
I. In the absence of Mg2+,
hUNG2 showed only a marginally elevated specificity
(kcat/Km) for ssU over dsU
(1.3-fold), whereas for hSMUG1 the corresponding ratio was 1.6. Thus
the common term "single-strand selective monofunctional uracil-DNA
glycosylase 1" could be justified for hSMUG1 under these conditions.
In the presence of Mg2+, however, this picture dramatically
changed as the ssU/dsU specificity ratio of hUNG2 increased ~40-fold.
This was caused by a 127-fold increase in
kcat/Km of hUNG2 against ssU,
whereas some 3-fold increase was observed with dsU. Interestingly, with
the dsDNA substrate Mg2+ appeared to influence the
catalytic turnover only. With the ssDNA substrate, however,
Mg2+ only affected the affinity (reduced
Km). Thus, the presence of physiological
concentrations of Mg2+ apparently turns hUNG2 into an
efficient, single-strand selective enzyme ideally suited for
identifying rare, deaminated cytosine residues residing in
single-stranded regions of DNA. A similar effect was not observed for
hSMUG1. Although the ssU/dsU specificity ratio increased from 1.6 to
3.8 (based on kcat/Km) in the
presence of Mg2+, this effect was solely caused by changes
in kcat. In fact, the presence of
Mg2+ reduced the affinity of hSMUG1 toward both
substrates.
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Table I
Kinetic constants of hUNG2 and hSMUG1 against
[3H]dUMP-containing calf thymus DNA and their modulation by
MgCl2 and APE1
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When both Mg2+ and APE1 were included in the assays, a
further increase in kcat/Km
was observed for hUNG2 against dsU, whereas
kcat/Km against ssU was
reduced by 50% compared with Mg2+ alone. However, hUNG2
still maintained a 7.4-fold preference for ssU over dsU. For hSMUG1,
the opposite effect was observed in the presence of APE1. The affinity
and turnover against both ssU and dsU increased, but the increase was
quantitatively higher with dsU. Thus, in the presence of both
Mg2+ and APE1, hSMUG1 displays a 2-fold preference for the
double-stranded substrate.
AP Sites Inhibit hSMUG1 but Not hUNG2--
AP sites have been
shown to be a strong (micromolar range) competitive inhibitor of the
catalytic hUNG domain, and this domain binds to AP sites more strongly
and more rapidly than to uracil-containing DNA (33). Such binding is
also observed with several other DNA glycosylases (36, 37). We
postulated that binding to the product AP site could be of crucial
importance to avoid mutagenic and cytotoxic effects of the AP site
until the subsequent AP endonuclease arrived and ensured further
processing of the damage site (33). Surprisingly, no inhibition by AP
sites was observed with the full-length hUNG2 protein when compared
with corresponding non-AP-containing oligonucleotides (Fig.
2A). An entirely different
effect was observed with hSMUG1. Whereas no specific inhibition was
observed with the AP-containing ss-oligonucleotides, a strong
inhibitory effect was observed with the AP-containing
ds-oligonucleotides (Fig. 2B). Furthermore, the AP:G
construct inhibited somewhat more strongly than AP:A. These results may
explain the 15-fold increase in
kcat/Km for hSMUG1 against
dsU when APE1 is present (Table I). Because APE1 does not stimulate
this activity in the absence of Mg2+ (Fig. 1C),
this likely occurs mainly by Mg2+-stimulated
endonucleolytic cleavage of the AP site, whereby hSMUG1 product
rebinding is blocked. hSMUG1 displacement from ds AP sites by APE1,
similar to human hTDG (36) may, however, be a contributing factor,
because a weak stimulation by APE1 is also observed in the absence of
Mg2+ (Fig. 1D) (note that APE1 binds to AP sites
in the absence of Mg2+, but cleaves at a very reduced
rate). Because single-stranded AP sites do not inhibit hSMUG1, such a
mechanism cannot explain the observed 2-fold increase in
kcat/Km of hSMUG1
against ssU by APE1. Active recruitment of hSMUG1 to the APE1·DNA or
formation of a hSMUG1·APE1 complex prior to substrate binding could
hypothetically contribute to this, but no such interaction has been
observed in immunoprecipitation experiments (23). It is also less
obvious why APE1 only stimulates the activity of hUNG2 against dsU.
Because hUNG2 is neither inhibited by AP sites in ssDNA nor dsDNA, and because the major effect of APE1 is on the Km
against both substrates, it is tempting to speculate that APE1 may
directly interact with hUNG2 and regulate its relative affinity against single-stranded versus double-stranded substrates.
Preliminary data from our laboratory indicate that direct APE1/UNG2
interactions in fact do occur in vitro, and that this
interaction depends on the phosphorylation patterns of hUNG2. This is
now under further investigation.
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Fig. 2.
Effects of apyrimidinic sites on hUNG2 and
hSMUG1. The activities of hUNG2 and hSMUG1 against
[3H]dUMP-containing calf thymus DNA were analyzed in the
presence of varying 19-mer oligonucleotides containing AP sites or
normal bases. Filled triangles, duplex AP:A;
filled inverted triangles, duplex
AP:G; filled circles, single-stranded AP
oligonucleotide; open inverted
triangles, C:G control; open circles,
single-stranded oligonucleotide containing C instead of an AP
site. A, hUNG2 in the absence of Mg2+.
B, hUNG2 in the presence of Mg2+.
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hSMUG1 Has Broader Substrate Specificity than hUNG2--
Boorstein
and co-workers (15) recently demonstrated that recombinant hSMUG1 and
SMUG1 purified from calf thymus were able to excise HmU from DNA
(HmU:A) in addition to U, although a direct comparison between the two
substrates was not performed. To investigate the relative efficiency of
hUNG2 and hSMUG1 against different uracil analogs, various
concentrations of both enzymes were incubated with a panel of
double-stranded oligonucleotides containing U:G, FU:A, HmU:G, C:G
(Fig. 3), and 5-OHU. The results
indicated narrow substrate specificity for hUNG2 restricted to uracil
and uracil analogs with minor structural modifications at the
5-position (substrate preference: U 
5-FU). In contrast, much
broader substrate specificity was observed for hSMUG1. In addition to
previously reported U and HmU, the enzyme was also active against FU
and C, with the order of preference U > HmU C > FU. Neither enzyme was able to excise 5-OHU under the present
conditions (data not shown). These results indicate considerable
structural differences in the active site of hSMUG1 compared
with hUNG2. Thus, whereas pyrimidines having bulky substitutions at the
5-position do not have access to the hUNG2 active site, hSMUG1 is able
to accommodate such substitutions at the 3-, 4-, and 5-positions.
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Fig. 3.
Substrate specificities of hUNG2 and
hSMUG1. Varying concentrations of hUNG2 or hSMUG1 were incubated
for 30 min at 37 °C with 5'-32P-labeled oligonucleotides
containing uracil, 5-FU, HmU, or C. 1 ng of APE1 was included in all
reactions. The upper bands observed after denaturing PAGE and
phosphorimaging represent uncleaved 19-mer substrate, whereas the lower
bands represent cleaved product. Extra bands observed below the APE1
cleavage products from FU and C are caused by some base loss and
cleavage during the final piperidine treatment.
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Based on analysis of UDG activities from Ung/ mice (23)
and the low Km value (0.035 µM) of
Xenopus SMUG1 (14), it was suggested that SMUG1 evolved to
counteract the mutagenic effects of deaminated cytosines, and that the
relative contribution of UNG2 to the repair of this lesion was reduced
in mammalian cells (23). The data presented in Fig. 3 indicate that
hUNG2 removes U from U:G mismatches even more efficiently than hSMUG1 and is thus a candidate enzyme to counteract cytosine deamination. To
further analyze this, the reaction kinetics of hUNG2 and hSMUG1 were
analyzed using oligonucleotides containing A, G, or no base opposite
the substrate (U of HmU) and in the presence of Mg2+ (Table
II). The results demonstrated that hUNG2
had a 300-fold higher kcat/Km
than hSMUG1 against U:G mismatches, and that this was caused both by a
lower Km and a higher kcat in
the case of hUNG2. Furthermore, hUNG2 had a >100-fold higher
kcat/Km than hSMUG1 against
ssU. Although the results with short oligonucleotides are not directly
comparable to the kinetic parameters obtained with
[3H]dUMP-containing calf thymus DNA (Table I), they
clearly demonstrate that both U:G and ssU are very good substrates for
hUNG2. The results furthermore indicate that HmU is a somewhat poorer
substrate for hSMUG1 than uracil, independent of the opposing base.
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Table II
Kinetic constants of hUNG2 and hSMUG1 against oligodeoxy nucleotide
substrates
The assays were performed in the presence of 7.5 mM
MgCl2 (final).
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hSMUG1 and hUNG2 Are Differently Localized within the
Nucleus--
We have previously demonstrated that hUNG2 is efficiently
translocated to nuclei and that, in the S-phase, it is clearly
accumulating in replication foci (4). This is in accordance with its
function in postreplicative removal of misincorporated uracil. Because the above analyses were performed with fixed cells, we wanted to
re-examine the translocation of hUNG2 in living cells. In cells transiently transfected with UNG2-EYFP (Fig.
4), a significant portion of hUNG2 was
localized outside replication foci. To ensure that this was not a
result of overexpression of the fusion protein to levels exceeding the
cellular translocation capacity, the fusion protein was stably
transfected under the control of the hUNG2 promoter. A similar result
was observed in these cells. hUNG2 appeared to be evenly distributed in
the nucleoplasm outside the S-phase, and a fraction of hUNG2
accumulated in replication foci in the S-phase. Furthermore, hUNG2
appeared to be excluded from nucleoli both in the S-phase and outside
the S-phase. hSMUG1, however, appeared to be less strictly localized to
nuclei, and a substantial fraction is distributed throughout the
cytoplasm. This was also the case when EYFP was fused to the C-terminal
end of hSMUG1 (data not shown). Furthermore, hSMUG1 appeared to be especially abundant in the nucleoli both in replicating and
non-replicating cells. The physical localization of the two proteins
indicates that both hUNG2 and hSMUG1 are available for repair of
deaminated cytosines outside replication foci, in accordance with their
substrate preferences. Only hUNG2, however, appears to have a function
in postreplicative repair in replication foci. Conversely, only hSMUG1 is observed in nucleoli. The latter observation is intriguing, and may
reflect a yet unrecognized function of hSMUG1 in nucleoli.
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Fig. 4.
Subnuclear localization of hUNG2 and
hSMUG1. HeLa cells were transiently or stably
(ProA-UNG2-EYFP) transfected with the indicated constructs, and
analyzed by laser confocal scanning microscopy as described under
"Experimental Procedures." The left column
shows subnuclear localization pattern of hUNG2 and hSMUG1 in fusion
with EYFP. Cells in early and middle (inset) S-phase can be
identified by the distinct focal distribution of PCNA
(middle column). Colocalization of hUNG2 and PCNA
is demonstrated in the merged pictures (right
column). Note that hUNG2 is largely excluded from nucleoli,
whereas hSMUG1 accumulates in nucleoli.
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hUNG2 Is the Major Glycosylase for Removal of Both Deaminated
Cytosine and Misincorporated Uracil in Nuclear Extracts--
Nilsen
et al. (23) recently provided evidence that SMUG1
represented a major glycosylase against U:G mismatches in ung-deficient mice. To analyze whether this was also so in human cells and to compare
the hSMUG1 activity against oligonucleotides containing U:A and U:G,
nuclear extracts from HaCaT, HeLa, and CX-1 cells were incubated with
neutralizing antibodies to specifically inhibit hUNG2,
hSMUG1, or both. In separate experiments the degree of inhibition and specificity of the anti-hUNG PU101 and antihSMUG1 PSM1 polyclonal antibodies were analyzed. Both antibodies proved to be
specific, and essentially no cross-reactivity was observed with the two
enzymes (Fig. 5A). The
apparently higher concentration of antibody necessary to inhibit hSMUG1
was a result of the relatively high concentration of hSMUG1 needed to
obtain measurable activity using [3H]dUMP-calf thymus DNA
as substrate. The latter was considered not to pose a problem when
neutralizing nuclear extracts, as the number of SMUG1 molecules per
cell in the present cell lines appears to be considerably lower than
UNG2. Whereas hUNG2 is easily detected by Western analysis of cell-free
extracts, we were not able to detect hSMUG1 without previous
concentration by immunoprecipitation (data not shown). Inhibition of
the recombinant proteins by the PBS2-encoded inhibitor Ugi was also
analyzed. Ugi proved to be a potent inhibitor of hUNG2. However,
~40% of the hSMUG1 activity was also inhibited at the concentrations
of Ugi needed to fully inhibit hUNG2 (data not shown). Thus, to provide
the highest specificity, neutralizing antibodies were used as
inhibitors throughout the present study. The inhibited nuclear extracts
were then incubated with 33P-labeled U141A and U141G. To
obtain measurable activities of hSMUG1, both the final concentration of
nuclear protein (0.33 µg/µl) and the incubation time (1 h) were
elevated. Even under these conditions, preincubation of the extracts
with hUNG2-neutralizing PU101 inhibited >90% of the total activity
against both substrates (Fig. 5B). This clearly indicates
that hUNG2 is the major activity against both misincorporated uracil
and deaminated cytosine in nuclear extracts, and supports our earlier
finding that UNG proteins represent >98% of the activity against
misincorporated uracil in HeLa total cell extracts (6). Furthermore,
essentially all UDG activity in the extracts could be inhibited by
preincubation with both PU101 and PSM1, indicating that hUNG2 and
hSMUG1 together represent essentially all detectable UDG activity in
human nuclei under these conditions.
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Fig. 5.
Inhibition by neutralizing antibodies
demonstrates relative contribution of hUNG2 and hSMUG1 activities in
nuclear extracts. A, recombinant hUNG2 or hSMUG1 were
preincubated with or without neutralizing anti-UNG (PU101)
or anti-SMUG1 (PSM1) prior to assay against
[3H]dUMP-labeled calf thymus DNA to monitor their
neutralizing capacity and specificity. Open
circles, hUNG2/PSM1; closed circles,
hUNG2/PU101; open squares, hSMUG1/PU101; closed
squares, hSMUG1/PSM1. B, 33P-labeled
19-mer oligonucleotides U141A and U141G (0.2 pmol) were incubated for
1 h at 37 °C with nuclear extracts (5 µg of protein) that
were preincubated in the absence or presence of neutralizing antibodies
(0.5 µg) as indicated. The lower bands represent 9-mer cleavage
products after uracil excision and piperidine cleavage.
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hSMUG1 Is the Major 5-Hydroxymethyluracil-DNA Glycosylase in
Nuclear Cell Extracts--
Several lines of evidence indicate that
human cells contain more than one HmU-DNA glycosylase (15, 38). To
quantitate the contribution of hSMUG1 to the total HmU glycosylase
activity in human nuclei, the CX-1 nuclear extract was incubated with a duplex oligonucleotide containing HmU:G, a substrate common to all
reported HmU-DNA glycosylases. Preincubation of the nuclear extract
with UNG-neutralizing PU101 did not affect the amount of excised HmU,
whereas preincubation with SMUG1-neutralizing PSM1 inhibited
essentially all HmU glycosylase activity (Fig. 6A). Thus, SMUG1 likely
represents the major HmU glycosylase activity in the nuclear extracts.
Alternatively, a HmU glycosylase antigenically similar to SMUG1 may be
present in human cells. To investigate the latter possibility, total
protein was extracted from synchronized HaCaT cells at different times
after release, and immunoprecipitated using PSM1 covalently linked to
magnetic Dynabeads (Fig. 6, B and C). SDS-PAGE
and Western analysis demonstrated a single band of similar molecular
mass as recombinant hSMUG1 in all the samples. This supports the theory
that hSMUG1 is the major HmU-DNA glycosylase in the cells. Furthermore,
the near constitutive expression of hSMUG1 in both quiescent cells and
through the cell cycle supports the idea that hSMUG1 is not linked to
the replication process. This is in contrast to the human UNG proteins,
which are markedly up-regulated in the S-phase (27, 39), and the
demonstrated function of hUNG2 in postreplicative removal of
misincorporated uracil.
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Fig. 6.
hSMUG1 is the major nuclear HmU-DNA
glycosylase and is constitutively expressed through the cell
cycle. A, 33P-labeled 19-mer
oligonucleotide HmU141G (0.4 pmol) was incubated for 2 h at
37 °C with CX-1 nuclear extract (13 µg of protein) that were
preincubated in the absence or presence of neutralizing antibodies (1.3 µg) as indicated. B, total cell extracts from HaCaT cells
were prepared from cells harvested at different time points after
release from serum starvation. hSMUG1 was immunoprecipitated using PSM1
covalently coupled to magnetic beads and subjected to SDS-PAGE and
Western analysis. The lane at the left contains
recombinant hSMUG. C, DNA synthesis in the cells harvested
at the same time points as in B were monitored by
[3H]thymidine pulse labeling.
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In Vitro Short-patch BER of U:A Is Initiated by hUNG2, whereas
Repair of U:G May Be Initiated by Alternative Mechanisms--
To
investigate whether UNG2 and SMUG1 from human cells were able to
initiate BER of uracil in vitro, nuclear extracts from HeLa
and CX-1 cells were preincubated with PU101 and/or PSM1 neutralizing antibodies prior to addition to an in vitro reconstituted
BER assay. As demonstrated in Fig.
7A, inhibition of nuclear
hUNG2 by PU101 completely abolished BER of the U:A plasmid substrate, whereas inhibition of hSMUG1 did not affect the amount of ligated product. This apparently reflected the initial rate of uracil removal
by the two enzymes, as demonstrated by excision of uracil from a 50-mer
U:A-containing oligonucleotide having a sequence identical to that of
the sequence flanking U:A in the BER substrate (Fig. 7B). In
this oligonucleotide the anti-UNG antibody completely inhibited uracil
excision. When a circular U:G plasmid was used as BER substrate,
preincubation with neutralizing antibodies had little effect on the
amount of repair product (Fig. 7A). This was entirely
unexpected, because uracil excision from the corresponding 50-mer U:G
oligonucleotide was completely inhibited by the anti-UNG antibody (Fig.
7B) under otherwise identical experimental conditions. In
all BER experiments above, at least 90% of the product resulted from
short-patch repair incorporation of only one nucleotide, whereas less
than 10% resulted from long-patch BER (data not shown). Thus, the
non-inhibited U:G repair was likely not caused by nucleotide excision
repair or mismatch repair. The latter was also verified by experiments
using nuclear extracts from mismatch repair-deficient HCT116 colorectal
cancer cells (ATCC CCL-247), in which U:G repair of the plasmid
substrate was also observed in the presence of both neutralizing
antibodies (data not shown). Moreover, the short-patch DNA-repair
process did not appear to be dependent on PCNA loading by replication
factor C, because exclusion of ATP from the reactions had no effect on
the steps prior to ligation (data not shown). A time-course BER
experiment indicated, however, that the degree of inhibition by the
neutralizing antibodies varied with the incubation time of the BER
reactions. At short incubation times, preincubation with anti-UNG
antibodies essentially abolished BER, whereas at prolonged incubation
times the level of BER product in the anti-UNG reactions approached
that of the uninhibited reactions (Fig. 7C). This might
indicate that repair of the U:G mismatch plasmid takes place by two
distinct short-patch BER mechanisms. In the initial phase of the
reaction, hUNG2-initiated short-patch BER dominates. In the late phase
initiation takes place by a yet unidentified mechanism, which is not
detected using U:G oligonucleotides as substrate. The possible
mechanisms of this alternative mode of repair will be discussed
below.
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Fig. 7.
hUNG2 but not hSMUG1 is a major initiator of
in vitro short-patch BER. A, nuclear
extracts from HeLa and CX-1 cells were assayed for short-patch BER
activity using a closed circular plasmid substrate containing a single
U:A or U:G at a defined position. The extracts were preincubated in the
presence or absence of neutralizing anti-UNG (PU101) and/or anti-SMUG1
(PSM1) as indicated and the BER reaction allowed to proceed for 60 min.
B, the degree of inhibition of the glycosylase step was
analyzed using 50 bp of 33P-labeled oligonucleotides
corresponding to the region encompassing uracil in the plasmid
substrates in A, and under otherwise identical conditions
except that dNTPs were replaced by ddCTP or ddTTP to arrest
polymerization and ligation. C, time-course BER assay using
CX-1 nuclear extracts. The extracts were preincubated with neutralizing
antibodies as in A, and reactions incubated for 10, 30, or
60 min. Ab, antibody.
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DISCUSSION |
By purifying the recombinant nuclear isoforms of hUNG2 and hSMUG1
to homogeneity, we are now, for the first time, able to carefully
determine the kinetic parameters of hUNG2 and hSMUG1 and to compare
their properties directly. The present biochemical data strongly
suggest that UNG2 has a broader function than mere postreplicative
removal misincorporated uracil, and likely is the major nuclear enzyme
for removal of deaminated cytosine in both double-stranded and
single-stranded DNA. Given the unsurpassed efficiency of hUNG2 compared
with hSMUG1 to remove uracil, it is reasonable to believe that SMUG1
serves another primary function in higher eukaryotes. hSMUG1 shares
many of the characteristics of hUNG2. There are, however, clear
differences, such as markedly lower turnover number, strong binding to
AP sites, broader substrate specificity, and the accumulation of
hSMUG1 in nucleoli but not in replication foci. Notably, the
activity of hSMUG1 against HmU is nearly as high as against U,
indicating that this might be the primary substrate for hSMUG1 in
vivo. This is also supported by the phylogenetic distribution of
SMUG1, which is linked to the use by organisms of 5-methylcytosine as a
mediator of gene expression (40). The present work shows that
recombinant hSMUG1 has a substrate preference for HmU in the order
ssHmU > HmU:G > HmU:A (Table II). For the corresponding
U-containing substrates, somewhat higher activities are observed,
although the order of preference is retained. This preference for
U-containing substrates is also found by Boorstein and co-workers (15).
More recently, however, a HmU-DNA glycosylase having distinct, but
overlapping, substrate specificity compared with hSMUG1 was
partially purified from HeLa cells (38). Like hSMUG1, this enzyme
excised both U and HmU opposite A and G, with a preference for U. Likewise, the enzyme had a weak activity against 5-fluorouracil.
However, no activity was observed against single-strand substrates or
3,N4-ethenocytosine. This activity may be the
same as previously observed by Radany et al. (41) in human
glioma cells, as the latter had no activity against G:T mismatches and
was strictly specific for paired uracil with a preference for U:G.
Interestingly, we identified a second EST derived from the SMUG1 gene
by data base search (NM_014311). This novel splice form is identical to
SMUG1 in the 135 N-terminal amino acids, but utilizes a different
reading frame of the SMUG1 3'-untranslated region to generate the 42 C-terminal amino acids. The biological function of this novel variant,
if any, is not known. Two of three main motifs believed to be important
for SMUG1 glycosylase activity (5), including the residues Asp-163 and Arg-243, are not present in the variant, suggesting that this protein
may have activities distinct from SMUG1.
The finding that hUNG2 is a highly effective single-strand selective
UDG in the presence of physiological concentrations of magnesium may
indicate that this is an important function of hUNG2 in
vivo. Transient single-strand regions occur frequently in
chromatin both as a consequence of "breathing" in A:T-rich DNA, and
in normal DNA metabolism such as replication, transcription, and
recombination. Furthermore, because the rate of deamination of cytosine
is more than 100-fold higher in ssDNA than in dsDNA (1), this would justify an efficient enzymatic scanning for uracil in ssDNA. This is
substantiated by the finding that the mutation rate in Ung-deficient yeast cells was increased 2-fold by a high transcription rate (42).
Efficient removal of deaminated cytosine from transcriptionally active
ssDNA could also be important to avoid miscoding transcripts and
synthesis of harmful proteins. The potential of generating such
proteins was demonstrated for human UNG, as a single base pair
substitution changed UNG from being a DNA repair enzyme to actually
becoming a mutator enzyme (12). Notably, hUNG2 contains two RPA-binding
motifs in the N-terminal regulatory domain (4), which may aid the
recruitment to single-stranded regions of DNA.
In the present study we identified a BER-initiating activity that is
apparently specific for long and/or circular U:G substrates, and that
is not inhibited by neutralizing antibodies against hUNG or hSMUG1.
Slow U:G-BER could in principle be initiated by the mismatch
glycosylases TDG or MBD4. However, in the cell lines used in this
study, hUNG2 and hSMUG1 represented all detectable UDG activity, even
at high concentrations of nuclear protein and at prolonged incubation
times. It is thus tempting to speculate that the observed repair is
initiated by a fraction of hUNG2 residing within a protein complex
specific for U:G mismatches, and that is not readily accessible to
binding by the antibodies. This is also supported by findings in our
laboratory that a fraction of UNG2 is not accessible for
immunoprecipitation. Interestingly, a 180-kDa multiprotein complex
isolated from bovine testis was able to perform complete BER of a
U:G-containing oligonucleotide (43). The complex contained pol ,
ligase I, AP endonuclease, and UDG, although the precise nature of the
latter two proteins was not determined. The mere size of such a
complex, if existent in human cells, may explain why the 50-mer U:G
oligonucleotide is not a substrate. The precise identification of the
components of such a complex must, however, await its isolation in
sufficient quantities for analysis by, e.g., mass spectrometry.
As shown in Fig. 8, uracil and HmU
in DNA may be present in different positions relative to a replication
fork, and in addition the sequence context may vary. It seems likely
that both the type of initiating UDG, as well as the BER subpathway in
the subsequent steps, will depend on these factors. Our data indicate
that the majority of HmU is excised by hSMUG1 outside of replication
foci, and is thus likely processed by short-patch BER. Deaminated
cytosine present in dsDNA prior to replication, e.g. in the
G1-phase, may in principle be removed by any of the four
identified human uracil-DNA glycosylases. Most likely, hTDG and hMBD4
mainly function in CpG contexts and in dsDNA only, whereas hSMUG1 and
hUNG2 may operate in any sequence context, albeit with varying
efficiency (6, 44, 45). SMUG1 was recently suggested to be a major
enzyme in repair of uracil in U:G mismatches (23). However, our data strongly suggest that, even in repair of U:G mispairs resulting from
cytosine deamination, hUNG2 may be a major player. hUNG2 has at least
as low a Km as hSMUG1, and is present in replication
foci as well as in the nucleoplasm. However, hUNG2 is essentially
excluded from nucleoli, whereas hSMUG1 accumulates in nucleoli,
suggesting that hSMUG1 may have a specialized role in uracil repair in
nucleoli. Among the uracil-DNA glycosylases, only hUNG2 specifically
accumulates in the replication foci during S-phase, and all
experimental evidence suggests that UNG2 has an important role in the
removal of misincorporated uracil in replication foci.
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Fig. 8.
Unified model for the occurrence and repair
of uracil and HmU in replicating and non-replicating chromatin.
The figure indicates the likely routes whereby uracil and HmU are
introduced in DNA. Note that, when deaminated cytosines occur in the
single-stranded region in front of the replication fork, hUNG2 probably
excises uracil and leaves an AP site that may stall the replication
fork and induce recombination or fork regression. Because AP
endonucleases are highly double-strand-specific, the risk of crating a
strand break (functionally a double-strand break) is very low. AP sites
at the replication fork are most likely repaired either by BER
subsequent to fork regression, or by recombination using information
from the sister chromatid (which is now double-strand in this short
region). Alternatively, AP sites are bypassed by translesion synthesis
(TLS).
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UNG2 is by far the most efficient enzyme for removal of uracil in
ssDNA. The lack of AP site rebinding by hUNG2 should not pose a problem
in ssDNA outside of replication foci because the double-helical DNA
conformation is likely restored prior to replication, thus creating a
substrate for APE1. Binding of APE1 then ensures that the downstream
processing steps take place by stepwise recruitment of BER-factors and
completion of repair prior to replication. One important problem is,
however, the following. How are deaminated cytosines that either escape
repair prior to replication, or is formed at the replication fork,
repaired, if at all? If uracil escapes repair and directs incorporation
of adenine, subsequent removal of uracil by hUNG2 and replacement by
thymine would result in a G:C A:T transition. This would be a poor
strategy unless postreplicative A:U pairs resulting from
misincorporation of A opposite a deaminated cytosine are processed
differently from misincorporated U opposite A. At present, there is no
experimental evidence supporting such differential postreplicative
processing. However, our data demonstrate that hUNG2 very efficiently
removes uracil from single-stranded DNA, and may thus generate an
abasic site that blocks replication. In addition, specific binding to RPA may boost recruitment of hUNG2 to single-stranded DNA in front of
the replication fork. The stalled replication fork may recruit proteins
required for fork regression and homologous recombination, which may be
alternative mechanisms to short-patch repair and long-patch repair in
the downstream steps subsequent to uracil removal. Fortunately, AP
endonucleases require double-stranded DNA so the risk of creating a
strand break (functionally a double-strand break) is small. Involvement
of recombination in the repair of abasic sites has been documented for
E. coli (46). Furthermore, induction of deamination of
cytosine by NO. is strongly cytotoxic in E. coli
recBCD cells deficient in recombination (47). Notably, disruption of
the genes for Ung and Fpg in these cells led to enhanced survival,
inferring that both Ung and Fpg create substrates for recombinational
repair (48). The finding that recombination factors are required for
processing of abasic sites in bacteria suggests that this may also be
the case in mammalian cells, because this basic process is highly
likely to be conserved. We therefore propose that uracil in
single-stranded DNA at the replication fork is incised by hUNG2 and
repaired by recombination or fork regression, which are both processes
requiring recombination proteins.
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ACKNOWLEDGEMENTS |
The pGFPCNAL2 construct was kindly provided
by Professors Heinrich Leonhardt and Christina M. Cardoso (Franz
Volhardt Clinic, Berlin, Germany). Recombinant hAPE1 was kindly
provided by Professor Ian D. Hickson (University of Oxford, Oxford,
United Kingdom). We thank Phoung Nguyen for preparing the stably
transfected cell lines expressing fluorescence-tagged UNG2. The
mismatch repair-deficient cell line HCT116 was kindly provided by Dr.
Jane Plumb, Department of Medical Oncology, University of Glasgow by
permission of Dr. Richard Boland, Department of Medicine and Cancer
Center, University of California.
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FOOTNOTES |
*
This work was sponsored by the Norwegian Cancer Society; the
Research Council of Norway; the Cancer Fund at St. Olavs Hospital, Trondheim, Norway; and the Svanhild and Arne Must Fund for Medical Research.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.
Present address: Cancer Research UK London Research Inst., Clare
Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
§
To whom correspondence should be addressed. Tel.:
47-73598693; Fax: 47-73598801; E-mail:
geir.slupphaug@medisin.ntnu.no.
Published, JBC Papers in Press, August 2, 2002, DOI 10.1074/jbc.M207107200
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ABBREVIATIONS |
The abbreviations used are:
UDG, uracil-DNA
glycosylase;
BER, base excision repair;
APE, AP endonuclease;
PCNA, proliferating cell nuclear antigen;
pol, polymerase;
EYFP, enhanced yellow fluorescent protein;
ECFP, enhanced cyan fluorescent
protein;
FU, fluorouracil;
C, 3,N4-ethenocytosine;
ss, single-stranded;
ds, double-stranded;
NLS, nuclear localization signal;
HmU, hydroxymethyluracil;
DTT, dithiothreitol;
EGFP, enhanced green
fluorescent protein;
BSA, bovine serum albumin;
5mC, 5-methylcytosine.
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REFERENCES |
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