[CANCER RESEARCH 47, 123-128, January I, 1987]
Biosynthesis of the Human Base Excision Repair Enzyme Uracil-DNA
Glycosylase1
Thomas M. Vollberg,2 Barbara L. Cool, and Michael A. Sirover
Fels Research Institute and the Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
ABSTRACT
The biosynthesis of the human DNA repair enzyme uracil-DNA
glycosylase has been characterized by the reaction of in vitro- and in
ciVo-produced protein with an anti-human placenta! uracil-DNA glyco
sylase monoclonal antibody. In vitro synthesis of the DNA repair enzyme
was examined after the translation of human placenta! polyadenylated
||iolv( \)*| RNA by immunoprecipitation of the |35S)methionine-labeled
translation products. As defined by sucrose density analysis, immunoprecipitable in rit m products were translated from 16S poly(A)* RNA and
1IS poly(A)* RNA. While the products of the 1IS poly(A)* RNA were
smaller than purified uracil-DNA glycosylase, the product of the 16 S
poly(A)* RNA had a molecular weight of 37,000, identical to the size
previously observed for purified human placenta! uracil-DNA glycosy
lase. Immunoblot analysis of human placenta! cell extracts and of normal
human fibroblast cell extracts demonstrated the recognition of one M,
37,000 protein. Immunoprecipitation of |35S|methionine-labeled normal
human cell extracts with the anti-glycosylase monoclonal antibody spe
cifically detected only the M, 37,000 uracil-DNA glycosylase protein.
Pulse-chase analysis demonstrated that the "S radioactivity in the M,
37,000 uracil-DNA glycosylase decreased over a 5-h interval. These
results show that immunoreactive human uracil-DNA glycosylase protein
was synthesized at its enzymatically active molecular weight of 37,000
as the primary translation product of a 16S polyadenylated messenger
RNA.
INTRODUCTION
Uracil can be formed in DNA either by the incorporation of
dUMP into DNA during DNA replication (1, 2) or by the
mutagenic deamination of cytosine residues (3,4). Uracil-DNA
glycosylase functions in the initial step of base excision repair
by catalyzing the degradation of the glycosyl bond between the
uracil base and its corresponding deoxyribose sugar moiety.
The resultant apyrimidinic site is a substrate for apurinic acid
endonuclease(s). Repair is completed by sequential catalysis by
subsequent enzymes within the base excision repair pathway
(5-7). Recent evidence suggests that eukaryotic cells enhance
base excision repair enzymes during the transition from the
quiescent to the proliferative state (8-15) and as the selective
regulation of excision repair genes in a manner which is intrinsic
to the cell cycle (16-18). The in vitro specific activity of uracil(8-11, 13-15), 3-methyladenine- (11, 12), and hypoxanthineDNA glycosylases (15) was increased in proliferating cells. In
synchronously growing cells, uracil-DNA glycosylase was reg
ulated in a distinct temporal sequence prior to the onset of
DNA replication (16-18). Further, hypermutable cells from
cancer-prone individuals with Bloom's syndrome were charac
terized by specific temporal alterations of uracil-DNA glyco
sylase regulation during the cell cycle (19).
To begin to examine the regulation of human DNA repair
genes at a molecular level we prepared a series of anti-uracilReceived 2/18/86; revised 7/24/86, 9/17/86; accepted 9/24/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by a grant to M. A. S. from the NIH (CA-29414)
and by grants to the Fels Research Institute from the NIH (CA-12227) and from
the American Cancer Society (SIG-6).
2 To whom requests for reprints should be addressed.
DNA glycosylase monoclonal antibodies using partially puri
fied human placenta! glycosylase as an antigen (20). Each
exhibited anti-uracil-DNA glycosylase activity as defined by
immunoprecipitation analysis. Three of the antibodies recog
nized antigenic determinants on homogeneous human placenta!
uracil-DNA glycosylase.3 Using one of these monoclonal anti
bodies we have characterized the in vitro synthesis of immuno
reactive uracil-DNA glycosylase protein from poly(A)+ RNA"
and its in vivo biosynthesis. We now report that immunoreactive
glycosylase protein was synthesized at a molecular weight of
37,000 from 16S poly(A)+ RNA. This molecular weight is
identical to that observed for catalytically active homogeneous
human placental uracil-DNA glycosylase. In addition, in vivo
biosynthesis as defined by immunoblot analysis of human fibro
blast cell sonicates and immunoprecipitation
of [-"SJmethionine-labeled cell protein failed to detect any precursor polypeptides. These results suggest that immunoreactive human uracilDNA glycosylase protein was synthesized at its catalytically
active molecular weight as the primary translation product of a
16S polyadenylated mRNA.
MATERIALS
AND METHODS
Cell Culture. Normal human skin fibroblasts, CRL 1222 (American
Type Culture Collection, Rockville, MD), were cultured in Dulbecco's
modified Eagle's medium (Hazleton Dutchland, Inc., Denver, PA)
supplemented with 10% fetal bovine serum, 2 mw L-glutamine, 100
Mg/ml streptomycin, and 100 units/ml penicillin at 37°Cin a humidified
atmosphere of 5% CO2 in air. For immunoblot analysis cells at subconfluent density (5 x IO6 cells/150-cm2 culture flask) were harvested by
trypsinization and washed twice in 50 ml of ice-cold sterile phosphatebuffered saline. The cells were then resuspended in 1.0 ml of ice-cold
Buffer I [20 mM Tris-HCl (pH 8.0), 1 mM diothiothreitol, and 20%
glycerol]. The cells were sonicated at 90 W for 30 s in an ice bath. The
sonicate was centrifuged at 300 x g for 10 min to remove any unbroken
cells. Protein concentration was determined by the method of Lowry et
al. (21).
RNA Isolation and in Vitro Translation. Placentas from normal
healthy births were obtained within 0.5 h of birth. Placental tissue,
dissected free of major vessels and membranes, was grossly minced
with scissors and forceps and then washed three times by suspension
in 500 ml of ice-cold 0.15 M potassium chloride and collected on sterile
gauze. The tissue was suspended in 300 ml of ice-cold 0.15 M potassium
chloride and pelleted by centrifugation at 300 x g for 10 min at 4°C.
The tissue (40 g) was homogenized in 100 ml of guanidium isothiocyanate buffer [6 M guanidium isothiocyanate, 5 mM sodium citrate
(pH 7.0), l MIM2-mercaptoethanol, and 0.5% SarkosylJ with a Brinkman polvi ron at the highest setting for 30 s. Cesium chloride was added
to the homogenate (1 g/2.5 ml of homogenate). RNA was pelleted
through a cushion of 5.7 M cesium chloride in 0.1 M EDTA (pH 7.5)
at 29,000 rpm in a Beckman SW40 rotor for 18-20 h at 20°C.
Poly(A)* RNA was isolated by 2 cycles of oligo(dT)-cellulose chromatography (22). Total placental RNA was heated to 65°Cfor 10 min
and then cooled to room temperature. The solution was adjusted to 0.5
M LiCl by the addition of an equal volume of 2x loading buffer [40 mM
3 G. Seal et al., submitted for publication.
4 The abbreviations used are: poly(A)* RNA, polyadenylated
sodium dodecyl sulfate; oligo(dT). oligodeoxythymidylate.
RNA; SDS,
123
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BIOSYNTHESIS
OF HUMAN
URACIL-DNA
GLYCOSYLASE
Tris-HCl (pH 7.6), 1.0 M lithium chloride, 1 HIM EDTA, and 1%
sodium dodecyl sulfate]. The RNA was applied to a 1.5-ml column of
oligo(dT)-cellulose (type 7; Pharmacia Molecular Biologicals, Piscataway, NJ) preequilibrated with loading buffer. The effluent was collected,
heated to 65°C,and reapplied to the column at room temperature. The
tive translation products were produced from the in vitro trans
lation of total placental RNA and placental poly(A)+ RNA
(results not shown). The largest of these translation products
had an apparent molecular weight equal to the M, 37,000 size
of enzymatically active uracil-DNA glycosylase in a homoge
column was washed with 10 ml of loading buffer, followed by 8 ml of neous preparation from human placenta.3 To determine the
loading buffer with 0.1 M lithium chloride. Poly(A)+ RNA was eluted
size of the uracil-DNA glycosylase mRNA, poIy(A)+ RNA was
with 6 ml of elution buffer [10 mM Tris-HCl (pH 7.5), 1 HIMEDTA,
and 0.05% sodium dodecyl sulfate]. The fractions containing .1:,1U sedimented through a 10-40% sucrose gradient. The RNA in
each fraction was precipitated and translated in vitro. Total
material were pooled and rechromatographed on a freshly equilibrated
oligo(dT)-cellulose column as described above. RNA was quantitated
nonspecific protein synthesis was detected by acid precipitation.
As shown in Fig. 1, one major peak of total poly(A)+ mRNAby absorbance at 260 nm (40 nu single stranded RNA/absorbance unit).
Translational activity of placenta! RNA preparations was assayed in
directed protein synthesis was detected. This 12S RNA presum
a reaction mix consisting of 40% rabbit reticulocyte lysates (Amersham
ably represented the most abundant placental mRNA species
Corp., Arlington Heights, IL), 2 nC\/ti\ assay volume [35S]methionine
which codes for human placental lactogen (25). Poly(A)+
(1000 Ci/mmol; Amersham Corp.), l unit,VI human placenta! RNase
mRNA-directed synthesis of human uracil-DNA glycosylase
inhibitor (Amersham Corp.), and magnesium acetate and potassium
was
then examined by immunoprecipitation
of the in vitro
acetate added to provide final cation concentrations of 1.1 and 120
mM, respectively. Translation reactions were incubated at 30°Cfor 60
translation mixtures with the 40.10.09 antibody (Fig. 1). In this
min. Translational activity was quantitated as the conversion of [35S] instance two distinct peaks of immunoprecipitable radioactivity
methionine to acid-insoluble radioactivity. Duplicate aliquots (2 n\
were detected. Each peak was clearly distinguished from the
major peak of total poly(A)+ RNA-directed protein synthesis.
each) of the reaction mixture were deacetylated in 0.5 ml of 1.0 M TrisNaOH (pH 10.7) for 10 min at 37°C.Acid-insoluble material was
The first peak corresponded to RNA with a sedimentation
precipitated with 2.0 ml of 25% trichloroacetic acid with 2% casamino
value of 16S. The second peak corresponded to RNA with a
acids at 4°Cfor 30 min. Acid-precipitable material was collected on
sedimentation value of IIS. No poly(A)+ mRNA-directed im
glass fiber filters, and radioactivity was determined spectrophotometrically.
Immunoprecipitation Analysis of |35S|Methionine-labeled Uracil-DNA
Glycosylase. Immunoprecipitations were performed as described by
Yamaguchi et al. (23). Cell free sonicates or in vitro translation mixtures
were diluted with an equal volume of ice-cold immunoprecipitation
Buffer A [100 mM Tris-HCl (pH 7.6), 0.6 M sodium chloride, 2 mM
EDTA, 2 mM phenylmethylsulfonyl fluoride, 2% Triton X-100, 1%
sodium deoxycholate, and 0.2% sodium dodecyl sulfate] and then
incubated overnight with either anti-human uracil-DNA glycosylase
monoclonal antibody (40.10.09) or spontaneously derived negative
control monoclonal antibody (1.05). Each antibody was added in a
volume of 100 n\ (60-70 /ig antibody protein). Monoclonal antibodies
were prepared from hybridoma supernatants by ammonium sulfate
precipitation and DEAE-cellulose chromatography as described previ
ously (20). The antigen-antibody complexes were precipitated by incu
bation with 100 n\ of rabbit anti-mouse IgG serum (Sigma Chemical
Co., St. Louis, MO). The precipitated complexes were collected by
centrifugation and washed four times by resuspension in 1.2 ml of icecold immunoprecipitation Buffer B [50 mM Tris-HCl (pH 7.6), 0.3 M
sodium chloride, 1 HIMEDTA, 1 mM phenylmethylsulfonyl fluoride,
1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate, and 0.3% unlabeled methionine] and recollection by centrifuga
tion. The immunoprecipitate was denatured in 50 n\ of electrophoresis
sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate,
10% glycerol, 700 mM 2-mercaptoethanoI, and 0.001% bromophenol
blue] for 30 min at room temperature followed by heating for 10 min
at 100'C.
Electrophoretic Analysis. Protein was analyzed by SDS-polyacrylamide gel electrophoresis (10% acrylamide) according to the method
of Laemmli (24). For analysis of [35S]methionine-labeled immunoprecipitates, the gels were fixed in 10% glacial acetic acid-45% ethanol,
then stained with 0.2% Coomassie Blue R-250, and destained in 7%
glacial acetic acid-20% ethanol. The destained gel was soaked for 30
min in Amplify autoradiographic enhancer (Amersham Corp.) and
dried under vacuum onto a paper backing. The migration of radioactively labeled material was detected by fluorography at -70°C with
Kodak X-OMAT AR-5 film (Eastman Kodak, Rochester, NY).
RESULTS
The anti-human uracil-DNA glycosylase monoclonal anti
body, 40.10.09, was used to identify uracil-DNA glycosylase
protein in in vitro translations of placental RNA. Preliminary
studies indicated that identical 40.10.09 antibody immunoreac-
munoprecipitable translational activity was detected for larger
mRNA species.
To examine the size of the immunoreactive protein synthe
sized by each mRNA, the 40.10.09 immunoprecipitates from
each translation reaction were analyzed by SDS-polyacrylamide
gel electrophoresis. As shown in Fig. 2, three distinct protein
products could be observed. The largest protein product (Mr
6H
23S
I
IBS I6S
11
I2S
3
1
Q I
Sp
ul 2
hi
I
O
(U
-2
4-
-I
Uj
Q
¡1
""
= 5
og
'O
K °•\5 A
kl
OC
£
o
2
9°
IO
15
20
FRACTION
Fig. 1. Sucrose gradient sedimentation of human uracil-DNA glycosylase
mRNA. A 10-40% linear sucrose gradient was formed in 10 ml by three successive
cycles of freezing at -70"C and thawing at 4'C of a sterile solution of 20% sucrose
in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 10 mM sodium chloride. Poly(A)+
RNA (27 ng) in 100 ¡A
sterile distilled water was heated at 65"C, cooled to room
temperature, and layered over the sucrose gradient. The RNA was sedimented by
centrifugation in a SW40 rotor (Smith Kline Beckman, Palo Alto, CA) at 37,000
rpm and 10°Cfor 17 h. Fractions (400 ¿il
each) were collected from the bottom
of the tube and RNA in the fractions was precipitated with yeast tRNA ( l S Mg;
BRL, Gaithersburg, MD) as carrier and 930 ¡A
ethanol at -20'C for >2 h. The
precipitate was collected by centrifugation, rinsed with 1.4 ml cold ethanol
(—20°C),
drained, and dried under vacuum. The RNA in each tube was dissolved
in 3.8 n\ sterile distilled water and translated in a 25-fil reaction mixture as
described in "Materials and Methods." Translational activity (•)was quantitated
as acid-insoluble 39Sradioactivity. Monoclonal antibody 40.10.09-immunoprecipitated material was heat denatured in SO /il of electrophoresis sample buffer and
the incorporation of [35S]methionine into antibody 40.10.09-immunoprecipitable
material (O) was quantitated spectrophotometrically by spotting a l-*il aliquot of
the denatured protein solution onto a glass fiber filter. Arrows, sedimentation of
standard RNA molecules in parallel gradients.
124
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BIOSYNTHESIS
OF HUMAN
URACIL-DNA
23S
Fig. 2. Electrophoretic analysis of immunoprecipitable translation products from sizefractionated poly(Ar RNA. Poly(A)* RNA
was size-fractionated and translated in vitro as
described in the legend to Fig. 1. The SDSdenatured antibody 40.10.09 immunoprecipitable material from each translation (in 49 n\)
was analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Lane numbers
correspond directly to the fractions of the su
crose gradient in Fig. 1. Arrows, sedimentation
of standard RNA molecules in parallel gra
dients, left ordinate, electrophoretic migration
of protein molecular weight standards; k, mo
lecular weight in thousands. The fluorogram
was exposed for 24 h at —70"C.
GLYCOSYLASE
I8S
IBS
I6S
I
I
I2S
69k —
45k —
30k —
14.3k —
I
23456
7
8
9
10 II 12 13 14 15 16 17 18 19 20 21 22
FRACTION
37,000) was precipitated from translations of the poly(A)+ RNA
from fractions 8 and 9 of the sucrose density gradient. This
corresponded to the 16S RNA fraction. The second protein
product (A/r 24,000) was synthesized from RNA in fractions
10-12 of the gradient. This corresponded to the US mRNA
fraction. In these same fractions (fractions 10 to 12) and in
fractions 13 and 14 a third immunoprecipitable protein product
(A/r 15,000) was seen. No immunoreactive protein with molec
ular weights greater than 37,000 was observed in any of the
translates. Only the 16S poly(A)+ RNA translated to yield a
product equal in size to homogeneous human uracil-DNA
glycosylase. The smaller immunoreactive translation products
were the products of shorter RNA molecules. Thus, immuno
reactive uracil-DNA glycosylase proteins appear to be the prod
uct of both a 16S and an 1IS poly(A)+ RNA.
The in vivo molecular weight of uracil-DNA glycosylase
protein was examined by immunoblot analysis of cell-free ex
tracts. As shown in Fig. 3, using a range of protein concentra
tions (42-170 Mg/lane), the 40.10.09 monoclonal antibody de
tected only one band of immunoreactive material in each sam
ple. The immunoreactive material migrated with an apparent
molecular weight of 37,000. Thus, the in vivo molecular weight
of immunoreactive species from crude human placenta! extracts
was identical to that of the purified placenta! uracil-DNA
glycosylase and to that of the full length in vitro translation
product. No immunoreactive material was observed at any
molecular weight other than 37,000. Similar results were ob
served using crude normal human fibroblast cell extracts (Fig.
4). This would suggest that immunoreactive protein was present
in vivo at a molecular weight comparable to that of the active
catalytic species. Further, no immunoreactive protein was pres
ent to a significant extent as a precursor or as an immunoreac
tive degradative intermediate.
The possibility that transiently produced precursor polypeptides might be formed in vivo was examined by the immunoprecipitation of uracil-DNA glycosylase from radiolabeled cells.
Normal human skin fibroblasts were labeled by incubation in
[35S]methionine for 24 h. Cell-free sonicates were immunoprecipitated with 1.05 or 40.10.09 monoclonal antibody. As shown
in Fig. 5/4, the 40.10.09 monoclonal antibody specifically pre
cipitated only one radiolabeled protein with an apparent molec
ular weight of 37,000. Several radiolabeled proteins of other
molecular weights were nonspecifically precipitated by both
monoclonal antibodies. The selective precipitation of the M,
37,000 immunoreactive protein is in accord with the immuno
blot analysis. However, this experimental procedure does not
— 66K
— 45K
—36K
—24K
Fig. 3. Immunoblot analysis of human placenta! protein. Crude human pla
centa! extracts were prepared by dissection and homogenizalion as described
previously (20). After SDS-gel electrophoresis, human placenta! protein was
electroblotted according to the method of Towbin et al. (42) onto 0.45-fim
nitrocellulose paper (BA 85; Schleicher and Scheull) in 5 mw Tris. 192 mM
glycine, 20% methanol, and 0.1% sodium dodecyl sulfate at 130 mA constant
current for 18 h. Uracil-DNA glycosylase in the portion of the nitrocellulose
paper corresponding to sample lanes of the gel was detected by using the antihuman uracil-DNA glycosylase mouse monoclonal antibody. 40.10.09, munii
noaffinity purified goat anti-mouse IgG, and mouse monoclonal peroxidaseantiperoxidase complexes (Sternberger-Meyer Immunochemicals, Inc., Jarrettsville, MD) in a modification of the immunoblot procedure of Johnson et al. (43)
as described previously.5 Goat anti-mouse IgG was preadsorbed with purified
human IgG (Sigma). Right ordinate, migration of protein molecular weight
markers, k, molecular weight in thousands. The limit of detection in the immu
noblot protocol is approximately 10 >»g
of crude placenta! extract. Lane I, partially
purified human placenta! uracil-DNA glycosylase (20 ng protein). Lanes 2, 3, and
4 correspond to 170, 85, and 42 ng of total placenta! protein applied to each
respective lane.
determine whether the M, 37,000 protein is assembled from
smaller molecules, such as those precipitated from the in vitro
translations, which might be only transiently present in their
unassembled form.
5 T. M. Vollberg et al., submitted for publication.
125
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BIOSYNTHESIS
OF HUMAN
URACIL-DNA
GLYCOSYLASE
result in the in vivo formation of immunoreactive degradative
intermediates with molecular weights of less than 37,000. Based
on these initial studies, further detailed analysis is required to
determine the proliferative dependent turnover of the uracilDNA glycosylase in human cells.
~~
66
K
—36K
—29K
—24K
Fig. 4. Immunoblot analysis of normal human fibroblast protein. Cell-free
sonicates of cultured normal human skin fibroblasts were prepared as described
in "Materials and Methods." After SDS-polyacrylamide gel electrophoresis, cell
sonicate protein (1-27 fig/lane) was electroblotted as described in the legend to
Fig. 3. Uracil-DNA glycosylase immunoreactive protein gel was detected by using
anti-human uracil-DNA glycosylase mouse monoclonal antibody (40.10.09) and
rabbit anti-mouse IgG serum. Righi ordinate, migration of protein molecular
weight markers; A, molecular weight in thousands; arrow, migration of purified
human placenta! uracil-DNA glycosylase in an adjacent lane of the gel. Lanes 1,
2, 3, and 4 correspond to 27.2, 10.9, S.S and I.I fig of total cell sonicate protein
applied to each respective lane. The limit of detection in the immunoblot protocol
is approximately 1 pg of crude fibroblast extract.
To investigate this possibility, cultured normal human cells
were pulse-labeled in [35S]methionine medium for 30 min.
Subsequently, cell sonicates were prepared either immediately
or after a 5-h chase period during which the cells were incubated
in unlabeled growth medium. 35S-labeled protein was immunoprecipitated with 1.05 or 40.10.09 antibody and analyzed by
SDS-polyacrylamide gel electrophoresis. If uracil-DNA glyco
sylase was assembled from transiently expressed immunoreac
tive precursor peptides, they would be detectable in the pulsed
samples. Subsequently, their distribution or intensity would be
altered in the pulse-chased samples. As shown in Fig. SB, only
the A/r 37,000 protein was specifically precipitated by the
40.10.09 antibody from both the pulsed cell sonicate and the
pulse-chased cell sonicate. After this short labeling interval, the
nonspecific immunoprecipitation
of a M, 46,000 protein was
observed using either control antibody 1.05 or monoclonal
antibody 40.10.09. Thus, immunoreactive uracil-DNA glyco
sylase precursor was not formed in normal human cells. Also,
in the pulse-chased cells there is visibly less radiolabel in the
M, 37,000 immunoreactive protein than was immunoprecipitable from cells which were pulsed but not chased. This indi
cated that there was significant metabolic turnover of the M,
37,000 uracil-DNA glycosylase in proliferating cells during the
5-h chase period. However, the turnover of this protein did not
DISCUSSION
Recent studies demonstrated
that eukaryotic excision repair
consists of a series of discrete enzymatic steps which comprise
singular biochemical pathways (5-7). Each pathway functions
to remove specific perturbations in DNA structure formed
spontaneously or after exposure to environmental agents (26).
Although the enzymology of eukaryotic excision repair has
been examined intensively, the mechanisms through which
human excision repair genes may be controlled remain unclear.
Previously, we demonstrated that the normal human cells reg
ulated the base excision repair pathway during cell proliferation
in a defined temporal sequence prior to the induction of DNA
replication in S phase (16-18). This regulation consisted not
only of the enhancement of the capacity of a cell to perform
base excision repair after exposure to specific DNA-damaging
agents but also of the enhancement of the in vitro specific
activity of base excision repair enzymes. In addition, we dem
onstrated that hypermutable cells from cancer-prone individ
uals could be characterized by specific defects in repair regula
tion which were correlated with their individual phenotypes
(19, 27). Thus, human cells may contain two excision repair
multigene families the expression of which would be dependent
on the proliferative state of the cell. Alternatively, normal
human cells may contain a single base excision repair gene
family the expression of which is increased during cell growth.
In either instance, hypermutable cells from cancer-prone indi
viduals may be characterized at the molecular level by altera
tions in such gene regulation.
To begin to examine this regulation at the molecular level we
examined the biosynthesis of a human DNA repair enzyme. In
particular, we determined whether the uracil-DNA glycosylase
was synthesized initially as a precursor polypeptide which re
quired a posttranslational cleavage to produce the active en
zyme. Immunoblot analysis using an anti-uracil-DNA glycosy
lase monoclonal antibody and normal human cell extracts dem
onstrated one single immunoreactive species with a molecular
weight of 37,000. This molecular weight is equivalent to that
observed for homogeneous human placenta! uracil-DNA gly
cosylase.3 No immunoreactive species of a higher molecular
weight was detectable even when normal human cells were
incubated with [35S]methionine for 24 h prior to harvesting and
immunoprecipitation. In contrast, the prokaryotic C^-methylguanine methyltransferase appears to be synthesized initially as
a polypeptide precursor (A/r 37,000). Subsequent cleavage re
sults in the production of a MT 18,000 active enzyme (28). It
remains unclear whether such processing is typical of prokar
yotic DNA repair enzymes or whether such posttranslational
cleavage relates to the regulatory role of the M, 37,000 protein
in the prokaryotic adaptive response.
Recent evidence demonstrates that prokaryotes contain a
single uracil-DNA glycosylase species. Each appears to be of
uniform size, with a molecular weight of approximately 18,00024,000 as defined by SDS-gel electrophoresis, gel filtration, or
glycerol gradient analysis (5). In contrast, there appear to be
conflicting reports on the number and size of eukaryotic base
excision repair enzymes. Molecular weight determinations for
the eukaryotic uracil-DNA glycosylase range from 19,000 to
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BIOSYNTHESIS
A.
..
OF HUMAN
URACIL-DNA
GLYCOSYLASE
B.
92.5K —
-92.5
69K —
—69K
46K —
—46K
^—
—»
30K —
K
—30K
1234
Fig. 5. | '''S|Mi-iIlumine radiolabeling of uracil-DNA glycosylase in cultured normal human skin fibroblasts. Normal human skin fibroblasts were cultured in 100mm culture dishes (Costar, Cambridge, MA) to a subconfluent density of 1.5 X IO6cells as described under "Materials and Methods." Approximately 3x10* cells
were used for each protocol. Cell sonicates were prepared and immunoprecipitations were carried out as detailed under "Materials and Methods." In A, cell protein
was labeled for 24 h with ["Sjmethionine (40 ¡iC\/m\,1000 Ci/mmol; Amersham Corp.) in methionine-free growth medium (methionine-free Dulbecco's modified
Eagle's medium, 10% dialyzed fetal bovine serum, 2 mm L-glutamine, 100 units penicillin, and 100 ng streptomycin per ml). The ["SJmethionine-labeled protein in
SO/iI of the cell sonicate was immunoprecipitated with mouse monoclonal antibody and rabbit anti-mouse IgG serum. The immunoprecipitated material was analyzed
by SDS-polyacrylamide gel electrophoresis and fluorography. Left ordinate, migration of protein molecular weight standards; K, molecular weight in thousands; arrow,
M, 37.000 40.10.09-immunoprecipitable
"S-labeled protein. Lane 1, immunoprecipitation with the negative control 1.05 mouse monoclonal antibody; Lane 2,
immunoprecipitation with anti-human uracil-DNA glycosylase 40.10.09 mouse monoclonal antibody. B, pulse-chase analysis. For the 30-min pulse, 10 ml of
methionine-free growth medium containing 60 nd/m\ [3!S]methionine (1000 Ci/mmol) were added to each culture dish. Cells were cultured for 30 min at 37"C. In
cultures which received a 5-h chase the ("SJmethionine medium was replaced after the labeling period. The "S-labeled cell protein in 100 M'of the cell sonicate was
immunoprecipitated with mouse monoclonal antibody and rabbit anti-mouse IgG serum as described under "Materials and Methods." Immunoprecipitated material
was heat denatured in 50 ^1 of electrophoresis sample buffer and then analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Right ordinate, migration
of protein molecular weight standards; arrow, M, 37.000 40.10.09-immunoprecipitable "S-labeled protein. In Lanes I and 2, cells were pulse-labeled for 30 min. Cell
sonicates were immunoprecipitated with the negative control 1.05 mouse monoclonal antibody (Lane I) or the anti-human uracil-DNA glycosylase 40.10.09 mouse
monoclonal antibody (Lane 2). In Lanes 3 and 4, the cells were pulse-labeled for 30 min and chased for 5 h. Cell sonicates were immunoprecipitated with the negative
control 1.05 mouse monoclonal antibody (Lane 3) or the anti-human uracil-DNA glycosylase 40.10.09 mouse monoclonal antibody (Lane 4).
50,000 (8, 29-33). Similarly, molecular weights for the 3methyladenine-DNA glycosylase range from 25,000 to 68,000
representing 1-3 species (34, 35). Multiple forms of partially
purified human placenta! apurinic acid endonucleases have been
reported (36), one of which may be absent in xeroderma pigmentosum cells (37). However, extensive purification from
human placenta resulted in the identification of a single placental apurinic acid endonuclease (38, 39). As defined by in vitro
translation of total RNA and poly(A)"1"RNA, we detected sev
eral immunoreactive human uracil-DNA glycosylase species.
Sucrose gradient density analysis demonstrated that each poly
peptide was encoded by separate mRNAs with sizes of 16S and
US, respectively. However, we were unable to detect the Mr
24,000 protein by immunoblot analysis of human placental cell
extracts or of normal human fibroblast cell extracts. Similarly,
this protein was not observed by immunoprecipitation
after
incubation of normal human cells with [35S]methionine for 24
h. Further, this protein was not detected after a pulse-chase
experiment. Thus, it would appear that the US mRNA is not
translated in vivo and presumably represented the in vitro trans
lation of a partially degraded mRNA. Alternatively, the IIS
RNA could encode for the mitochondria! uracil-DNA glycosy
lase (40). The results presented in this report demonstrated a
single 16S size class for the uracil-DNA glycosylase mRNA
and one size class of immunoreactive protein(s) in vivo. This
mRNA size class is similar to that reported by Legerski et al.
(41) for the 13S and 14S mRNAs which encode the products
required to complement the nucleotide excision repair capacity
of xeroderma pigmentosum cells, complemention groups D and
E, respectively. Further molecular analysis is required to ex
amine whether the size class of glycosylase mRNA which codes
for the Mr 37,000 protein(s) includes multiple mRNAs tran
scribed from distinct uracil-DNA glycosylase structural genes.
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Biosynthesis of the Human Base Excision Repair Enzyme
Uracil-DNA Glycosylase
Thomas M. Vollberg, Barbara L. Cool and Michael A. Sirover
Cancer Res 1987;47:123-128.
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