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(Received for publication, August 5,
1994; and in revised form, October 26, 1994) From the
The G:U mismatch in genomic DNA mainly arises from deamination
of cytosine residues and is repaired by the base excision repair
pathway. We found that a bovine testis crude nuclear extract conducts
uracil-initiated base excision repair in vitro. A 51-base pair
synthetic DNA substrate containing a single G:U mismatch was used, and
incorporation of dCMP during repair was exclusively to replace uracil.
A neutralizing polyclonal antibody against DNA polymerase During the life span of any organism, genomic DNA can be damaged
by various physical or chemical agents, and for faithful reproduction,
all or most of these damaged DNA sites must be repaired. Organisms have
complex systems for repairing DNA lesions including the following:
direct lesion removal, recombination, base excision repair,
methylation-directed mismatch repair, and nucleotide excision repair of
bulky
adducts(1, 2, 3, 4, 5) . In
cases where DNA repair is carried out by the base excision repair (BER) ( Uracil arises in DNA
by two independent pathways: first, deamination of cytosine to uracil
occurs spontaneously or in response to oxidizing chemical agents such
as sodium bisulfite (3) and nitric oxide(23) , giving
rise to G:U mismatch. Second, higher levels of dUTP in the cell are
associated with incorporation of dUMP into DNA opposite a template A.
In the case of the G:U mismatch, there is mutagenic potential if the
mismatch is not corrected, leading to the G:C
Figure 1:
A, sequences of duplex oligonucleotide
substrates. Lower strand (LS) contained dUMP at position 22,
relative to the 5` end G at position 1. Substrate containing dCMP in
place of dUMP served as a reference. Restriction endonuclease sites,
along with upper (US) and LS designations, are indicated. The
last nucleotide (position 51 or 3` end) of the lower strand was ddATP
in all substrates. Four other oligonucleotide substrates (not shown)
were prepared using the identical sequence except that G:T, A:T, A:C,
or A:U base pairs were created at position 22 where T, T, C, and U were
in the lower strand, respectively (see Fig. 3). B, a
composite figure showing uracil base excision repair assay with bovine
testis nuclear extract. The repair reaction was carried out as
described under ``Experimental Procedures.'' Autoradiograms
of typical gel electrophoresis results are shown. PanelA, time course of product accumulation for the standard
repair reaction. Aliquots were withdrawn at various times of incubation
as indicated above each lane. Panel B, specificity of
nucleotide incorporation in the repair reaction. The repair reaction
was carried out with substrate containing a G:C or G:U bp at position
22 (A) and [
Figure 3:
Substrate specificity for the incision
steps of the repair reaction. Experiments were conducted as described
under ``Experimental Procedures,'' and an autoradiogram of
typical results is shown. Different substrates containing
5`-end-labeled lower strand were incubated in the standard repair
reaction devoid of dNTP and an ATP-regenerating system (ATP,
phosphocreatine, and creatine phosphokinase). The base pair
corresponding to position 22 is shown at the bottom of each lane, and
the substrates are illustrated at the top, as well as the position of
the 5`-
To establish the position of [
Figure 2:
A composite figure showing reaction
requirements for base excision repair. The repair assay was carried out
as described under ``Experimental Procedures.''
Autoradiograms of typical results are shown. A, modifications.
As shown at the top of each lane, the repair reaction was conducted
without any modification, in the absence of MgCl
As omitting ATP probably blocked the repair reaction at
the DNA ligase step, we decided to exploit this property to identify
products of uracil DNA glycosylase-endonuclease activities, as well as
the DNA polymerase activity step. The lower strand of the
G:U-containing duplex substrate was 5`-end-labeled, and this substrate
was incubated with the extract without other additions, i.e. the reaction mixture did not contain dCTP, ATP, or ATP
regenerating systems. Under these conditions, labeled material
corresponding to the starting 51-nucleotide molecule was shifted to an
oligonucleotide 21 residues long. When dCTP was then added to the
reaction mixture, we observed a band corresponding to an
oligonucleotide of 22 residues, as shown in Fig. 2B. To
determine if the lack of ATP would result in accumulation of the 22
nucleotide long intermediate, we carried out a similar reaction (in the
presence of all components) using 5`-end-labeled lower strand of the
G:U substrate. As shown in Fig. 2C, in the absence of
dCTP, a 21-nucleotide product accumulated that was converted to
full-length product in the full repair reaction containing dCTP. When
ddCTP was used in place of dCTP, accumulation of a 22-nucleotide
product was observed, as expected for a
Figure 4:
A composite figure showing inhibition of
the repair reaction. A, as indicated at the top of each lane,
the standard repair reaction was carried out in the presence of bovine
testis nuclear extract alone (none) or in the presence of a 100-fold
molar excess of ddCTP over dCTP or 5 µg/ml aphidicolin.
Additionally, the nuclear extract was mixed (1:1 volume) with preimmune
IgG or polyclonal antibody (IgG) raised against purified rat
Figure 5:
A,
fractionation of the base excision repair reaction proteins by gel
filtration column chromatography. As described under
``Experimental Procedures,'' approximately 100 mg of bovine
testis crude nuclear extract was loaded onto Mono Q and Mono S columns
connected in tandem. The Mono S column was eluted with 1 M KCl, and the proteins were concentrated in Centricon-10
concentration units. A, absorbance (280
Fractions also
were assayed for overall G:U repair activity. Product accumulation,
corresponding to the 22-residue molecule, but not the fully repaired
51-residue molecule, was maximal in fraction 32. Additionally, the
original sample applied to the Superose 12 column was found to have
base excision repair activity (lane S) as expected. Overall,
these results show that several of the enzymatic activities required
for the repair reaction could be separated from one another by gel
filtration chromatography and that all of the enzymatic activities
required were
Figure 6:
Reconstitution of the repair reaction and
its inhibition by
Figure 7:
Reconstitution of the repair reaction with
different DNA polymerases. A, fraction number 36 of the
Superose 12 gel filtration column was used to conduct the repair
reaction. Purified DNA polymerases
To establish that the DNA polymerases
used in the reactions were active, we carried out a primer extension
assay under conditions identical to those used for the repair reaction.
As shown in Fig. 7B, all the polymerases tested showed
abundant activity, but the amount of
Figure 8:
Uracil base excision repair reaction in S. cerevisiae. A, reconstitution of the repair
reaction was carried out as described in Fig. 6except that 0.1
µg of purified S. cerevisiae
Next, we
prepared nuclear extract from the S. cerevisiae strain
LP3041-6D (wild type) and the strain JWY355 carrying a Studies to understand the DNA polymerase requirement(s) for
various eukaryotic excision repair mechanisms have been under way for a
number of years and have led to the understanding that for gap-filling
DNA synthesis, the process can be loosely categorized by repair gap or
patch size as follows(30) : long-patch repair (>50 nt) as in
mismatch repair; intermediate-patch repair (24-50 nt) as in
nucleotide excision repair; and short-patch repair ( Our results with the S. cerevisiae BER in vitro system corroborate the
results of Wang et al.(14) . We conclude there was no
apparent requirement for In conclusion, we
have demonstrated by several criteria that the uracil-initiated BER DNA
polymerase activity in our crude extract is Addendum-DNA
polymerase
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 949-957
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conducts the Gap-filling Step in Uracil-initiated Base
Excision Repair in a Bovine Testis Nuclear Extract (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(-pol) inhibited the repair reaction. ddCTP also inhibited the
repair reaction, whereas aphidicolin had no significant effect,
suggesting that activity of -pol was required. Next, the base
excision repair system was reconstituted using partially purified
components. Several of the enzymatic activities required were resolved,
such that DNA ligase and the uracil-DNA
glycosylase/apurinic/apyrimidinic endonuclease activities were
separated from the DNA polymerase requirement. We found that purified
-pol could restore full DNA repair activity to the DNA
polymerase-depleted fraction, whereas purified DNA polymerases 
,
, and
could not. These results with purified proteins
corroborated results obtained with the crude extract and indicate that
-pol is responsible for the single-nucleotide gap filling reaction
involved in this in vitro base excision repair system.
)pathway, a damaged or inappropriate base is excised from
DNA and replaced by the base pair complementary nucleotide. DNA
synthesis to replace the nucleotide involves incorporation of only one
or a few dNMP residues (6, 7) . In mammalian systems,
information on the role of any one of the five cellular DNA polymerases
in DNA repair has been largely restricted to evidence obtained from
inhibitor studies(8) . Since the DNA polymerase inhibitors,
such as aphidicolin, dideoxynucleoside, N-ethylmaleimide, or
antibodies, can inhibit more than one DNA polymerase, interpretations
on involvement of a DNA polymerase in any one DNA repair mechanism have
generally been confounded(8) . The present study was designed,
first, to unequivocally determine whether there is a role for -pol
in mammalian BER, second, to develop a system in which mammalian BER
can be reconstituted from purified proteins, and third, to test the
performance of other DNA polymerases in mammalian BER in
vitro. Wiebauer and Jiricny (9) demonstrated involvement
of -pol in a G:T-initiated base excision repair reaction by HeLa
nuclear extract, using both anti--pol antibody and
dideoxynucleotide inhibition. Matsumoto and Bogenhagen (10) argued that -pol may be responsible for gap filling
during repair of a tetrahydrofuran lesion by BER in a Xenopus
laevis oocyte extract, and more recently Dianov et
al.(11) , using a human cell nuclear extract system for
uracil-initiated BER, obtained inhibition by dideoxynucleotide; this
led these workers to conclude that -pol was responsible for the
DNA synthesis step. Although these studies pointed to a role of
-pol in short-patch DNA repair, more specific studies were
required to settle the question of -pol involvement because of the
following considerations. The polymerase requirement in the
lymphoblastoid cell line-based BER system studied by Dianov et al.(11) was not clear cut. First, the reaction was inhibited
by the -pol inhibitor aphidicolin and only partially blocked by
the -pol inhibitor ddNTP, yet purified mammalian -pol is not
inhibited by 100 µg/ml aphidicolin and is >95% inhibited by
ddNTP at a ddNTP/dNTP ratio of only 10(12, 13) .
Further, it is known that -pol is inhibited by ddNTP, at a high
ratio of ddNTP to dNTP and can exhibit only partial inhibition by
aphidicolin(13) . Thus, the inhibition pattern of the BER
system studied by Dianov et al.(11) tends to confound
the interpretation that -pol was involved. Second, the -pol
requirement for the HeLa extract G:T-initiated BER system described by
Wiebauer and Jiricny (9) was assigned with a -pol antibody
that is non-neutralizing and relatively low titer. Although this
antibody, under appropriate conditions, can specifically recognize
-pol in a crude extract, use of a high titer, -pol-specific,
neutralizing antibody would strengthen the conclusion that -pol is
included in BER. Third, the picture concerning -pol involvement in
BER was further complicated recently by the discovery of a
-pol-like DNA polymerase in the yeast Saccharomyces cerevisiae along with findings by Wang et al.(14) . These
workers developed a S. cerevisiae in vitro system for
uracil-initiated BER and found that repair synthesis for osmium
tetroxide and UV-damaged DNA was conducted by DNA polymerase .
This finding is interesting in light of the presence of a
-pol
like enzyme in S. cerevisiae (DNA polymerase IV) and the
observation that a deletion strain for the corresponding gene has no
apparent phenotype(15, 16) . Hence, the studies with
the S. cerevisiae system provided no indication of a -pol
involvement in BER, and Wang et al.(14) have
suggested that results on yeast DNA polymerase requirements in DNA
repair may be extrapolated to mammalian DNA repair. In light of these
ambiguities and apparent contradictions, and earlier demonstrations
that purified -pol can completely fill short gaps in
vitro(17, 18, 19) , we undertook a study
to further examine the putative role of -pol in uracil-initiated
base excision repair. To approach the question, we developed a crude
nuclear extract-based in vitro BER system and two neutralizing
polyclonal antibodies to -pol. We chose uracil-initiated repair as
our model for BER because it is a well documented pathway in eukaryotic
cells, and several of the mammalian enzymes likely to be involved are
available as recombinant proteins, including uracil-DNA glycosylase,
apurinic/apyrimidinic (AP) endonuclease, and DNA ligase I (for reviews,
see (20, 21, 22) ).A:T transition
mutation. To establish an in vitro system, we used a synthetic
oligonucleotide-containing uracil at a defined position, along with
appropriate sites for restriction enzyme analysis of products. Our
system is based on a bovine testis crude nuclear extract (24) .
This in vitro system promotes robust base excision repair to
convert the G:U mismatch to G:C. Our results indicate that DNA
polymerase
is solely responsible for the single nucleotide
gap-filling synthesis in uracil-initiated BER in bovine testis nuclear
extracts but not in S. cerevisiae.
Materials
Radiolabeled nucleotides were from DuPont NEN or ICN
Radiochemicals. Electrophoresis grade acrylamide and bisacrylamide were
from Bio-Rad. dNTPs, ATP, dideoxy-CTP (ddCTP), bovine serum albumin, T4
polynucleotide kinase, and restriction endonucleases were from
Boehringer Mannheim. Formamide and urea were from Life Technologies,
Inc. Human DNA polymerase was from Molecular Biology Resources,
Inc. Human placental PCNA and DNA polymerases and
were a
generous gifts from Dr. M. Lee, University of Miami, FL. Recombinant
human and rat DNA polymerases
were purified as reported earlier (25, 26) . M13mp18(+) ssDNA and the M13 universal
sequencing primer were from Pharmacia Biotech Inc. Purified synthetic
oligodeoxynucleotide primers complementary to various regions of the
multicloning site of M13mp18 ssDNA (and synthetic template) were from
Genosys Biotechnologies, Inc. S. cerevisiae strains were
kindly provided by Dr. L. Prakash.Buffers
Homogenization buffer A was 10 mM HEPES, pH 8.0, 1.5
mM MgCl
, 10 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1
µg/ml pepstatin A, and 10 mM sodium metabisulfite. Buffer
B was 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1
µg/ml pepstatin A, and 10 mM sodium metabisulfite, while
buffer C is buffer B with 5% (v/v) glycerol.Nuclear Extract Fractionation
Nuclear Extract
Frozen bovine testis was from J.
Schmidt Co. (Baltimore, MD) or Pel-Freeze Biological (Rogers, AR) and
held at -80 °C until use. Nuclear extract was prepared as
reported earlier(24) . The final fraction, after 40%
(NH
)SO precipitation and dialysis
against buffer B, is referred to as nuclear extract (Fraction I) and
was stored at -80 °C until further use.Mono Q and Mono S Ion-exchange Chromatography
Mono
Q and Mono S ion-exchange columns (1 
10 cm) were attached in
tandem and equilibrated with buffer C. These columns were run at a
constant flow rate of 10 ml/h using a LKB pump. Approximately 15 ml
(
100 mg of protein) of nuclear extract (Fraction I) was loaded on
the column where it first passed through Mono Q and then over the Mono
S ion-exchanger. The columns were washed with two to three column
volumes of buffer C. The two columns were then separated, and the Mono
S column was eluted with buffer C containing 1 M KCl. The
fractions containing most of the eluted proteins were dialyzed
overnight against 100 volumes of buffer C, centrifuged at 12,000
g for 5 min to remove any insoluble material, and then
concentrated with a Centricon-10 (Amicon) at 3,000 g to the desired volume (Fraction II).Gel Filtration Column Chromatography
Fraction II,
200 µl, was loaded on a LKB FPLC Superose 12 HR 10/30 column
pre-equilibrated with Buffer C containing 1 M KCl, and the
column was run at a flow rate of 0.25 ml/min. Fractions (0.5 ml) were
concentrated with Centricon-10 concentration units and washed with
buffer C to remove salt, and concentrated fractions were stored at
-80 °C until further use. To standardize the column, 40
µl of Bio-Rad gel filtration molecular weight marker standard
mixture containing thyroglobulin, bovine -globulin, chicken
ovalbumin, equine myoglobin, and vitamin B-12 (670,000-1,350 kDa)
was diluted to 200 µl in buffer C with 1 M KCl. The
standard solution, 200 µl, was loaded on a FPLC Superose 12 column
and run under similar condition, and 0.5-ml fractions were collected.
Base Excision Repair and Product Analysis
The reaction conditions for base excision repair by bovine
testis nuclear extract were similar to those reported
earlier(11) . Standard reaction mixtures (50 µl) contained
100 mM Tris-HCl, pH 7.5, 5 mM MgCl, 1
mM dithiothreitol, 0.1 mM EDTA, 2 mM ATP,
0.5 mM NAD, three deoxynucleoside 5`-triphosphates at 20
µM each, 5 mM diTris-phosphocreatine, 10 units of
creatine phosphokinase, 40 nM of duplex oligonucleotide,
2-20 µM of the fourth
[-P]dNTP (specific activity
10
-10
disintegrations/min/pmol) and 50
µg (protein) of crude nuclear extract. Reactions were incubated for
10 min at 37 °C and stopped by addition of EDTA and NaCl to final
concentrations of 50 mM and 0.3 M, respectively. The
DNA was extracted with phenol-chloroform and precipitated with three
volumes of non-chilled ethanol. The DNA precipitate was collected
immediately by centrifugation to minimize co-precipitation of dNTPs,
dried under vacuum, and resuspended in 10 µl of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. The duplex oligonucleotide
(0.1-0.2 pmol) was incubated for 1 h at 37 °C with
20-50 units of restriction endonuclease under the conditions
recommended by the manufacturer. Unless otherwise specified, reactions
were carried out in the presence of 40 µCi of
[-P]dCTP (3000 Ci/mmol) alone, and the
reaction was stopped by adding an equal volume of ``stopping
solution'' (40 mM EDTA and 80% formamide) to the reaction
mixture. After incubation at 95 °C for 2 min, the DNA was separated
by electrophoresis on a 12% polyacrylamide gel containing 7 M urea in 89 mM Tris, 89 mM boric acid, and 2
mM EDTA (pH 8.8). Gels were fixed, dried, and autoradiographed
to visualize the reaction products.
-Pol Assays-Pol assays were conducted as described
earlier(19) . M13mp18(+) ssDNA was used as template and
M13 universal sequencing primer (17 nt) along with 51-mer synthetic
primer were annealed to create a 5-nt gapped substrate.Preparation of Antibodies
Antisera specific for intact rat -pol and its ssDNA
binding 8 kDa domain (27) were raised by immunization of
rabbits as described (28) . Recombinant rat -pol and 8 kDa
domain were purified as reported previously(26) . Specific
antibodies to -pol and 8 kDa domain were purified by affinity
chromatography, using Epoxy-activated Sepharose 6-B gel (Pharmacia)
coupled with -pol or 8 kDa domain. Coupling of the ligand and
subsequent elution of the bound antibodies from the gel were conducted
as suggested by the manufacturer. Antigenic and chemical
characterizations were performed using appropriate dilutions of the
affinity-purified antibodies to -pol or 8 kDa domain. These
affinity-purified polyclonal antibodies to -pol and 8 kDa domain
are referred to as anti--pol and anti-8 kDa, respectively. The IgG
fraction of the preimmune serum was purified by protein A-Sepharose
Cl-6B (Pharmacia) column.SDS-Polyacrylamide Gel Electrophoresis and Immunoblot
Analysis
SDS-polyacrylamide gel electrophoresis was performed
according to the method of Laemmli(29) , using 9.5%
discontinuous slab gels. The proteins were transferred onto a
nitrocellulose membrane (Bio-Rad) in a transblot apparatus (Bio-Rad),
according to the manufacturer's instructions. Subsequently, the
nitrocellular membrane was probed with affinity-purified anti--pol
(1:10,000) or preimmune IgG. Goat anti-rabbit IgG conjugated to
horseradish peroxidase (Bio-Rad) at 1:10,000 dilution was used as the
secondary antibody and was detected with an ECL chemiluminescence
system (DuPont).Uracil Base Excision Repair in S. cerevisiae
Nuclear extract was prepared from the S. cerevisiae wild type strain LP3041-6D (MAT leu 2-3 leu 2-112 trp 1 
ura 3-52)
and the isogenic -pol gene deletion strain JWY355 (MAT leu 2-3 leu 2-112 trp ura 3-52 -pol ::URA 3) exactly as described by
Wang et al.(14) . The base excision repair reaction
was carried out as with testis nuclear extracts.
Base Excision Repair with the Bovine Testis Nuclear
Extract
To establish an in vitro system for base
excision repair, we surveyed various sources of extract and substrate
molecules. The bovine testis nuclear extract was selected because it
has abundant uracil-initiated BER activity as will be described below
and therefore will be an excellent starting fraction for purification
of repair proteins. We used a synthetic oligonucleotide molecule
designed so that we could readily study single nucleotide gap filling
during repair. Incorporation of [-P]dCMP
during the BER reaction was carried out on a 51-residue duplex
oligonucleotide substrate containing a U residue at position 22 in the
lower strand (Fig. 1A). An autoradiogram illustrating
the time course of the repair reaction is shown in Fig. 1B, panel A. Radiolabel corresponding to
the 51-nucleotide product molecule appeared as a discrete band after 2
min of incubation, and an increase in product was seen with increasing
time of incubation. At the longest interval, some product bands shorter
than 51 residues appeared, which may have been the result of nuclease
activity in the nuclear extract. We adjusted the concentration of DNA
substrate and time of incubation so that the product formed
corresponded to no more than 25% of the DNA substrate in the reaction
mixture. We first examined the questions of whether this incorporation
of [
P]dCMP was specific to the G:U mismatch and
if other dNTPs could substitute for dCTP. As shown in Fig. 1B, panel B, the repair reaction was
carried out separately in the presence of different
-P-labeled dNTPs using either G:U or G:C containing
duplex (51 bp) oligonucleotide substrates, differing only at position
22 in the lower strand. Incorporation of radiolabel into full-length
product molecules occurred only with the substrate carrying the G:U
mismatch, and [
P]dCMP was the only nucleotide
incorporated among those tested (Fig. 1B panel B).
P]dNTP (*) as indicated
above each lane. Panel C, position of the
-P-label incorporated nucleotide in the
51-oligonucleotide product. The repair reaction was conducted in the
presence of 10-20 units of various restriction endonucleases, as
shown at the top of each lane, except for AccI and XbaI, where the reaction product was phenol extracted and
ethanol precipitated before subjecting it to restriction digestion. Numbers on the right show the lower strand size (nts) of the
fragment generated by restriction enzyme analysis. The illustration at
the top shows the position of [
P]dCMP in the
different restriction fragments and their respective position and size
in reference to the substrate.
P(*) label.
P]dCMP
incorporation in the 51-residue product, we carried out restriction
endonuclease digestion of the reaction product. In the experiment shown
in Fig. 1Bpanel C, BamHI, PstI, or SalI was added at the end of the reaction,
while in the cases of AccI or XbaI, the repair
reaction product was purified before subjecting it to digestion.
Different labeled fragment sizes were generated by the action of each
endonuclease. Based on the digestion pattern, it was evident that
[
P]dCMP was in the 5-residue region between the XbaI and AccI restriction sites (Fig. 1A), presumably at position 22 in the lower
strand. The labeled product molecule was not altered by treatment with
uracil-DNA glycosylase or heating at 70 °C, as expected (data not
shown).
Characterization of the Base Excision Repair
Reaction
The repair reaction with the G:U-containing substrate
was further characterized by modifying the reaction conditions. As
shown in Fig. 2A, eliminating MgCl completely abolished repair activity; some accumulation of a
22-nucleotide intermediate product was observed in the absence of ATP
and ATP regenerating system (phosphocreatine and creatine
phosphokinase), but minimal amounts of ligated 51 nucleotide product
were found; addition of NaCl to 200 mM greatly reduced the
activity.
or ATP
regenerating system (ATP, phosphocreatine, and creatine phosphokinase),
and in the presence of 200 mM NaCl. B, analysis of
intermediate products formed during the repair reaction. The
5`-end-labeled lower strand containing unlabeled dUMP was incubated in
the standard repair reaction devoid of dNTPs and ATP-regenerating
system (ATP, phosphocreatine, and creatine phosphokinase). The reaction
was carried out for 20 min in the presence and absence of dCTP as
shown. C, effect of addition of dideoxy-CTP in the repair
reaction. The repair reaction was carried out in the presence of
5`-end-labeled substrate. The lower strand, containing the unlabeled
dUMP residue, carried the 5`-end label. The standard repair reaction
was carried out in the absence or presence of dCTP and ddCTP, as shown
at the top of each lane. The number in the right-hand margin
indicates the length (nts) of the radiolabeled products. Whereas NE at the top represents bovine testis nuclear
extract.
-pol-mediated reaction.Specificity of the Excision Reaction
We found that
a molecule migrating in the gel as a 21-nucleotide oligomer is an
intermediate in the overall repair reaction. This intermediate could be
readily observed by conducting the reaction in the absence of dCTP. To
examine whether the phosphodiester backbone incision reaction producing
this 21-nucleotide intermediate was specific to a uracil-containing
substrate, we designed different combinations of base pairs at position
22 by altering the lower and upper strand of the 51-bp oligonucleotide (Fig. 1A), i.e. A:C, A:U, A:T, G:C, G:U, and
G:T. The lower strand of all these substrates was 5`-end-labeled.
Reactions were carried out in the absence of dCTP, ATP, and ATP
regenerating systems (phosphocreatine and creatine phosphokinase). As
shown in Fig. 3, use of only the A:U- and G:U-containing
substrates gave rise to the intermediate product molecule of 21
nucleotides, indicating that our repair reaction was specific for the
uracil base. The absence of the intermediate product of 21 nt in the
case of G:T and other mismatch substrates may be attributed to
relatively short incubation times used for uracil-initiated BER, thus,
distinguishing this reaction from that of G:T mismatch repair reaction
described by Wiebauer and Jiricny(9) . Use of substrates with a
5`-end-labeled upper strand, instead of lower strand, failed to result
in any shorter products.Inhibition of the Repair Reaction
Results of Fig. 2and Fig. 3indicated that the uracil base was
selectively removed and replaced by the base complimentary to the
template G, and then the strand was sealed by DNA ligase to generate
the full-length 51-bp product. The fact that ddCMP was incorporated and
accumulated suggested that -pol was involved in the repair
reaction. To initially examine the DNA polymerase requirement, we used
the inhibitors ddCTP and aphidicolin. As shown in Fig. 4, ddCTP
completely inhibited the repair reaction, whereas aphidicolin did not
have a noticeable effect (in an experiment not shown, neutralizing
antibodies against -pol did not inhibit the repair reaction),
suggesting that -pol, but not -pol was involved. We also
carried out the repair reaction by preincubating the nuclear extract
separately with preimmune serum or with polyclonal antibodies raised
against native -pol and its 8 kDa domain, respectively. Preimmune
serum had no effect, whereas the antibodies raised against -pol or
the 8 kDa domain completely inhibited the repair reaction (Fig. 4A). These two polyclonal antibodies, which were
raised and purified in the current study, gave a positive signal on
immunoblotting with purified -pol, bovine testis nuclear extract,
and S. cerevisiae DNA pol IV, a homolog of -pol (S.
cerevisiae -pol); these antibodies did not cross-react with
PCNA, human DNA polymerases, , , and
, or with E.
coli pol I (Fig. 4B). When preincubated with
purified
-pol, both of these new antibodies completely inhibited
-pol enzymatic activity (Fig. 4C) but did not
alter activity of the other DNA polymerases (data not shown).
-pol
or its 8 kDa domain. The proteins were preincubated at 0-1 °C
for 45 min. The standard repair reaction as indicated was carried out.
The position of the product is indicated by arrow. B,
characterization of the anti--pol polyclonal antibodies. Western
blot of S. cerevisiae -pol (2 µg) Escherichia
coli pol I (0.5 µg), PCNA (2 µg), -pol (2 µg),
-pol (2 µg),
-pol (0.5 µg), rat -pol (150
ng), and bovine testis nuclear extract (150 µg) was
conducted using preimmune serum and antibodies (as shown above) as
described under ``Experimental Procedures.'' C, the
effect of preimmune serum and antibodies were tested on -pol gap
filling activity, as described under ``Experimental
Procedures'' and indicated at the top of each
lane.
Separation of DNA Repair Activities on Superose 12 FPLC
Column
Bovine testis crude nuclear extract was passed though
Mono Q and Mono S columns connected in tandem. The Mono S column was
separated and eluted with 1 M KCl, and the eluate was
concentrated before loading it onto a Superose 12 gel filtration column
pre-equilibrated with buffer containing 1 M KCl to minimize
protein-protein interaction (complex formation). The protein elution
profile of the Superose 12 column is shown in Fig. 5A.
Individual fractions were subjected to various enzymatic assays. DNA
ligase activity eluted near the void volume and peaked in fraction 28
as judged by the formation of 73-mer ligase product (Fig. 5B). -Pol was the next activity to emerge
from the column with maximum 5-nucleotide gap-filling activity in
fraction 31, corresponding to the 39-kDa -pol monomer. To monitor
uracil DNA glycosylase/AP endonuclease activities, the duplex substrate
was 5`-end-labeled in the uracil-containing strand, and the reaction
was carried out in the absence of dCTP, ATP, phosphocreatine, and
creatine phosphokinase. Accumulation of a 21-residue product was
maximal in fraction 33 (Fig. 5B).
) elution
profile of FPLC Superose 12 HR 10/30 gel filtration column. A sample,
200 µl (
300 µg of protein) of concentrated 1 M KCl
fraction of Mono S column, was injected onto Superose S 12 column, and
0.5-ml fractions were collected. Respective positions of the molecular
weight markers are indicated in the figure. B, assay of
different enzymatic activities in the column fractions. All the
fractions were concentrated to 100 µl in buffer C. An equal
volume (10 µl) of fraction sample was used in each enzymatic
activity assay. DNA ligase and DNA polymerase activities were
determined as described under ``Experimental Procedures.''
The DNA ligase end product (73-mer) resulted after ligation of the
-pol gap filling reaction product (22-mer) with 51 nt oligo
annealed down stream to the M13 primer to create a 5-nt gap.
Glycosylase and endonuclease activities were detected by using a G:U
containing substrate with 5`-end-labeled U-containing strand. The
standard repair reaction was carried out in the absence of dNTP, ATP,
phosphocreatine, and creatine phosphokinase. A standard repair assay
was also conducted on the Mono S column fraction (lane S) that
was loaded on to the Superose 12 column. Enzymatic activities, their
respective products, and the fractions tested are shown in the
figure.
50 kDa, except for DNA ligase.Reconstitution of Base Excision Repair Reaction
We
used fraction 36 (Fig. 5B) to reconstitute the repair
reaction, as this fraction had uracil DNA glycosylase/AP endonuclease
activities, but was devoid of overall repair, DNA polymerase, or DNA
ligase activities. As shown in Fig. 6, fraction 36 alone failed
to show any overall DNA repair activity, and the addition of T4 DNA
ligase had no effect. However, the usual 22-residue intermediate
product molecule was formed when purified -pol was added.
Formation of this product was not observed when the 31 kDa domain of
-pol was added instead of intact -pol (data not shown). Next,
we found that the 22-residue product was shifted to the full-length
product (51-mer), when both -pol and DNA ligase were added.
Formation of the 22-residue intermediate product by fraction 32 was
abolished by polyclonal antibody against -pol or its 8 kDa domain.
Finally, the presence of -pol in fraction 32 was confirmed by
immunoblotting; this experiment confirmed the presence of the 39-kDa
enzyme (data not shown).
-pol antibodies. Fraction numbers 36 and 32 of
the Superose 12 gel filtration column were assayed for base excision
repair. T4 DNA ligase (3 µg), purified recombinant -pol (0.4
µg), and both T4 ligase and -pol together were added in the
repair reaction with fraction 36, while only ligase was with fraction
32. Inhibition of the repair activity was tested by addition of
fraction 32 preincubated on ice (1:1 volume) with preimmune serum and
the polyclonal antibodies raised against -pol and its 8-kDa domain
as shown in the figure.
Reconstitution of the Base Excision Reactions with
Different DNA Polymerases
Our results indicated that purified
-pol can reconstitute the BER reaction in vitro and that
the endogenous polymerase in fraction 32 is -pol, as the enzyme is
a 39-kDa DNA polymerase inhibited by specific anti--pol
antibodies. To determine if other purified mammalian DNA polymerases
were capable of conducting a single nucleotide gap filling reaction, we
studied highly purified human DNA polymerases , , and
using fraction 36, as in Fig. 6. We found that only
-pol
could reconstitute the full BER reaction; a very minor activity was
observed for DNA polymerase , in the presence of a large excess of
enzyme (Fig. 7A), and no activity was found with
polymerases and
.
(0.4 µg), (1.5
µg), (0.89 µg), and
(2.9 µg) were used as
indicated in the figure. Polymerase S activity was measured in the
presence of 3 µg of PCNA. B, primer extension activity of
the various DNA polymerases were tested by annealing M13mp18 (+)
ssDNA template and 17-mer universal primer as shown in the figure.
Identical reaction conditions and polymerase concentrations were used
as above except that 40 µM unlabeled dATP, dGTP, and dTTP
were included.
-pol used in these
experiments was much less than for the other polymerases. Although the
amount of polymerases used was different, a comparison of the
ratio/repair activity to primer extension activity, indicated that
-pol is far more active in the base excision repair reaction than
is -pol.Uracil Base Excision Repair in S. cerevisiae
A
study of uracil and osmium tetroxide-initiated BER in vitro in S. cerevisiae extract implicated DNA polymerase , and the
reaction could be influenced by DNA polymerases
and
(14) . Since the presence of
-pol-like enzyme was
recently demonstrated in S.
cerevisiae(15, 16) , we tested the role of
purified S. cerevisiae -pol in our mammalian BER system.
As shown in Fig. 8A, purified S. cerevisiae -pol was able to reconstitute polymerase activity in fraction
36 (as in Fig. 6), and the catalytic activity was neutralized by
anti-rat -pol antibody; this was expected as anti-rat -pol
antibody cross-reacts with S. cerevisiae -pol and also
inhibits its gap-filling activity (data not shown).
-pol was used. Equal
volumes of S. cerevisiae -pol and anti-rat -pol
antibody were preincubated before carrying out the repair reaction as
shown in the figure. B, the repair reaction was carried out as
described under ``Experimental Procedures.'' Nuclear extract
(25 µg) from either wild type strain or -pol gene deletion
strain was used. Nuclear extract from wild type strain was preincubated
with equal volume of preimmune and anti-rat -pol antibody as shown
in the figure.
-pol
gene deletion, as described under ``Experimental
Procedures.'' The uracil-initiated BER reaction, as reported by
Wang et al. (14) , was conducted with nuclear extract
from both strains (Fig. 8B). In both cases, the
22-residue intermediate product and a small amount of the completely
repaired 51-residue product were formed. Most of the 22-mer product
could be converted to 51-mer produced by addition of T4 DNA ligase
(data not shown), suggesting either a low level of DNA ligase or
partial inhibition of ligase activity in the nuclear extracts.
Formation of these products was not inhibited by anti--pol
antibodies. In an experiment not shown, product formation was not
inhibited by ddNTP. Further, addition of purified S. cerevisiae -pol had only marginal affect (data not shown). These results
corroborate the results of Wang et al.(14) and
suggest a different DNA polymerase requirement for uracil-initiated BER
in S. cerevisiae than in mammalian cells.
3-4 nt)
as in base excision repair. Two studies of BER in vitro,
involving short-patch repair (9, 11) , have pointed to
a requirement for -pol, and the enzyme has been clearly
demonstrated to have the capacity to conduct short-patch gap filling in vitro(17, 18, 19) . Our results
established that -pol is responsible for the single-nucleotide DNA
synthesis step involved. Multiple observations indicated that the DNA
strand containing uracil was repaired via a single-nucleotide excision
gap: 1) dCTP alone was sufficient to support the reaction; 2) the
product of the endogenous endonuclease incision step was only one
nucleotide shorter (i.e. 21 nt) than the position of uracil
residue; 3) addition of pure -pol extended the 21-nt intermediate
product molecule by only one nucleotide; 4) the 22-nucleotide
intermediate product accumulated in the reaction mixture when ATP was
omitted to intentionally block the activity of DNA ligase; 5) and
finally, the DNA synthesis reaction was completely blocked by two
specific, neutralizing polyclonal antibodies to -pol. Four of the
major enzymatic activities required for the reaction, DNA ligase, DNA
polymerase, uracil-DNA glycosylase, and endonuclease(s), could be
partially resolved by gel filtration chromatography using a Superose-12
column. DNA ligase activity eluted at a higher molecular mass than the
other activities, all of which had molecular mass of 50 kDa. Once
ligase activity had been removed, addition of the DNA ligase-containing
fraction to the depleted extract only partially restored the BER
activity (data not shown). The requirement for DNA ligase, however,
could be fully complemented by addition of purified T4 DNA ligase. In a
similar fashion, activities providing uracil DNA glycosylase and
endonuclease (processing of the AP site) could be purified free of the
40-kDa DNA polymerase activity (i.e.
-pol), and
addition of purified -pol was capable of restoring full repair
activity. Furthermore, among the four different purified mammalian DNA
polymerases tested here, DNA polymerase was the only enzyme
capable of restoring full repair activity. In these experiments with
polymerase-depleted fractions, DNA polymerase was able to
minimally reconstitute activity but this required a very high enzyme
concentration. Neither aphidicolin nor neutralizing antibody to
-pol had a blocking effect with the crude extract system. In a
related study (data not shown), addition of purified DNA polymerase
to HeLa cell nuclear extract resulted in 5-10-fold increase
in the uracil-initiated BER whereas DNA polymerase , , and
did not, suggesting a role for
-pol.-pol, since extract from the -pol
deletion strain was fully active in BER. In addition, the S.
cerevisiae BER reaction was not inhibited by ddNTP or neutralizing
antibody to -pol. These results indicate that the S.
cerevisiae and mammalian systems for BER under study here appear
to have different DNA polymerase requirements.-pol. These criteria
include: its small size, inhibition of activity by neutralizing
polyclonal antibodies against -pol, complete inhibition by
dideoxynucleotide, and by reconstitution of the BER activity using
purified -pol. Interestingly, a recent study (31) indicating embryonic lethality caused by a null mutation
of the -pol gene, along with the results of Wang et al.(32) and Sadakane et al.(33) showing
novel alterations in -pol mRNA in colorectal cancers and in Werner
syndrome cells, respectively, raise the possibility of a role of
-pol in these diseases. The multiple enzymatic activities in the
lower molecular weight fractions from our gel filtration column, which
contain uracil-DNA glycosylase and endonuclease, have not been
resolved. There is not yet a consensus in the literature, as to the
involvement of AP endonucleases alone, AP endonuclease and 5`3`
exonuclease, AP endonuclease and deoxyribophosphodiesterase (dRpase),
or other cellular protein (for discussion, see (11) ), together
with uracil-DNA glycosylase to create the single nucleotide gap.
)
We are indebted to Dr. M. Lee for providing purified
PCNA and DNA polymerase and
, and we thank Drs. L. Prakash,
S. Lloyd, S. Mitra, and B. Van Houten for their valuable suggestions
and critical reading of the manuscript.
is able to conduct repair of only natural AP sites in
abasic site repair in X. laevis oocytes(34) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. S. DeMott, S. Zigman, and R. A. Bambara Replication Protein A Stimulates Long Patch DNA Base Excision Repair J. Biol. Chem., October 16, 1998; 273(42): 27492 - 27498. [Abstract] [Full Text] [PDF]
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R. J. Sanderson and D. W. Mosbaugh Fidelity and Mutational Specificity of Uracil-initiated Base Excision DNA Repair Synthesis in Human Glioblastoma Cell Extracts J. Biol. Chem., September 18, 1998; 273(38): 24822 - 24831. [Abstract] [Full Text] [PDF]
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D. K. Srivastava, B. J. Vande Berg, R. Prasad, J. T. Molina, W. A. Beard, A. E. Tomkinson, and S. H. Wilson Mammalian Abasic Site Base Excision Repair. IDENTIFICATION OF THE REACTION SEQUENCE AND RATE-DETERMINING STEPS J. Biol. Chem., August 14, 1998; 273(33): 21203 - 21209. [Abstract] [Full Text] [PDF]
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R. Prasad, W. A. Beard, P. R. Strauss, and S. H. Wilson Human DNA Polymerase beta Deoxyribose Phosphate Lyase. SUBSTRATE SPECIFICITY AND CATALYTIC MECHANISM J. Biol. Chem., June 12, 1998; 273(24): 15263 - 15270. [Abstract] [Full Text] [PDF]
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R. Prasad, W. A. Beard, J. Y. Chyan, M. W. Maciejewski, G. P. Mullen, and S. H. Wilson Functional Analysis of the Amino-terminal 8-kDa Domain of DNA Polymerase beta as Revealed by Site-directed Mutagenesis. DNA BINDING AND 5'-DEOXYRIBOSE PHOSPHATE LYASE ACTIVITIES J. Biol. Chem., May 1, 1998; 273(18): 11121 - 11126. [Abstract] [Full Text] [PDF]
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W. A. Deutsch, A. Yacoub, P. Jaruga, T. H. Zastawny, and M. Dizdaroglu Characterization and Mechanism of Action of Drosophila Ribosomal Protein S3 DNA Glycosylase Activity for the Removal of Oxidatively Damaged DNA Bases J. Biol. Chem., December 26, 1997; 272(52): 32857 - 32860. [Abstract] [Full Text] [PDF]
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D. M. Wilson III and L. H. Thompson Life without DNA repair PNAS, November 25, 1997; 94(24): 12754 - 12757. [Full Text] [PDF]
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B. P. Engelward, G. Weeda, M. D. Wyatt, J. L. M. Broekhof, J. de Wit, I. Donker, J. M. Allan, B. Gold, J. H. J. Hoeijmakers, and L. D. Samson Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase PNAS, November 25, 1997; 94(24): 13087 - 13092. [Abstract] [Full Text] [PDF]
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A. M. Chagovetz, J. B. Sweasy, and B. D. Preston Increased Activity and Fidelity of DNA Polymerase beta on Single-nucleotide Gapped DNA J. Biol. Chem., October 31, 1997; 272(44): 27501 - 27504. [Abstract] [Full Text] [PDF]
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Z. Wang, X. Wu, and E. C. Friedberg Molecular Mechanism of Base Excision Repair of Uracil-containing DNA in Yeast Cell-free Extracts J. Biol. Chem., September 19, 1997; 272(38): 24064 - 24071. [Abstract] [Full Text] [PDF]
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M. F. Hashim, N. Schnetz-Boutaud, and L. J. Marnett Replication of Template-Primers Containing Propanodeoxyguanosine by DNA Polymerase beta . INDUCTION OF BASE PAIR SUBSTITUTION AND FRAMESHIFT MUTATIONS BY TEMPLATE SLIPPAGE AND DEOXYNUCLEOSIDE TRIPHOSPHATE STABILIZATION J. Biol. Chem., August 8, 1997; 272(32): 20205 - 20212. [Abstract] [Full Text] [PDF]
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R. A. O. Bennett, D. M. Wilson III, D. Wong, and B. Demple Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway PNAS, July 8, 1997; 94(14): 7166 - 7169. [Abstract] [Full Text] [PDF]
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P. R. Strauss, W. A. Beard, T. A. Patterson, and S. H. Wilson Substrate Binding by Human Apurinic/Apyrimidinic Endonuclease Indicates a Briggs-Haldane Mechanism J. Biol. Chem., January 10, 1997; 272(2): 1302 - 1307. [Abstract] [Full Text] [PDF]
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R. J. Sanderson and D. W. Mosbaugh Identification of Specific Carboxyl Groups on Uracil-DNA Glycosylase Inhibitor Protein That Are Required for Activity J. Biol. Chem., November 15, 1996; 271(46): 29170 - 29181. [Abstract] [Full Text] [PDF]
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J. Singh, L. Su, and E. T. Snow Replication across O6-Methylguanine by Human DNA Polymerase beta in Vitro. INSIGHTS INTO THE FUTILE CYTOTOXIC REPAIR AND MUTAGENESIS OF O6-METHYLGUANINE J. Biol. Chem., November 8, 1996; 271(45): 28391 - 28398. [Abstract] [Full Text] [PDF]
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O. I. Lavrik, R. Prasad, W. A. Beard, I. V. Safronov, M. I. Dobrikov, D. K. Srivastava, G. V. Shishkin, T. G. Wood, and S. H. Wilson dNTP Binding to HIV-1 Reverse Transcriptase and Mammalian DNA Polymerase beta as Revealed by Affinity Labeling with a Photoreactive dNTP Analog J. Biol. Chem., September 6, 1996; 271(36): 21891 - 21897. [Abstract] [Full Text] [PDF]
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S. Narayan, F. He, and SamuelH. Wilson Activation of the Human DNA Polymerase beta Promoter by a DNA-alkylating Agent through Induced Phosphorylation of cAMP Response Element-binding Protein-1 J. Biol. Chem., August 2, 1996; 271(31): 18508 - 18513. [Abstract] [Full Text] [PDF]
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C. E. Piersen, R. Prasad, S. H. Wilson, and R. S. Lloyd Evidence for an Imino Intermediate in the DNA Polymerase beta Deoxyribose Phosphate Excision Reaction J. Biol. Chem., July 26, 1996; 271(30): 17811 - 17815. [Abstract] [Full Text] [PDF]
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R. Prasad, R. K. Singhal, D. K. Srivastava, J. T. Molina, A. E. Tomkinson, and S. H. Wilson Specific Interaction of DNA Polymerase beta and DNA Ligase I in a Multiprotein Base Excision Repair Complex from Bovine Testis J. Biol. Chem., July 5, 1996; 271(27): 16000 - 16007. [Abstract] [Full Text] [PDF]
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N. Oda, J. K. Saxena, T. M. Jenkins, R. Prasad, S. H. Wilson, and E. J. Ackerman DNA Polymerases alpha and beta Are Required for DNA Repair in an Efficient Nuclear Extract from Xenopus Oocytes J. Biol. Chem., June 7, 1996; 271(23): 13816 - 13820. [Abstract] [Full Text] [PDF]
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W. A. Beard, W. P. Osheroff, R. Prasad, M. R. Sawaya, M. Jaju, T. G. Wood, J. Kraut, T. A. Kunkel, and S. H. Wilson Enzyme-DNA Interactions Required for Efficient Nucleotide Incorporation and Discrimination in Human DNA Polymerase beta J. Biol. Chem., May 24, 1996; 271(21): 12141 - 12144. [Abstract] [Full Text] [PDF]
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G. Frosina, P. Fortini, O. Rossi, F. Carrozzino, G. Raspaglio, L. S. Cox, D. P. Lane, A. Abbondandolo, and E. Dogliotti Two Pathways for Base Excision Repair in Mammalian Cells J. Biol. Chem., April 19, 1996; 271(16): 9573 - 9578. [Abstract] [Full Text] [PDF]
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 Y Matsumoto and K Kim Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair Science, August 4, 1995; 269(5224): 699 - 702. [Abstract] [PDF]
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D. K. Srivastava, T. Y. Rawson, S. D. Showalter, and S. H. Wilson Phorbol Ester Abrogates Up-regulation of DNA Polymerase [IMAGE] by DNA-alkylating Agents in Chinese Hamster Ovary Cells J. Biol. Chem., July 7, 1995; 270(27): 16402 - 16408. [Abstract] [Full Text] [PDF]
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M. J. Jezewska, S. Rajendran, and W. Bujalowski Energetics and Specificity of Rat DNA Polymerase beta Interactions with Template-primer and Gapped DNA Substrates J. Biol. Chem., May 4, 2001; 276(19): 16123 - 16136. [Abstract] [Full Text] [PDF]
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O. I. Lavrik, R. Prasad, R. W. Sobol, J. K. Horton, E. J. Ackerman, and S. H. Wilson Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate. EVIDENCE FOR THE ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 IN DNA REPAIR J. Biol. Chem., June 29, 2001; 276(27): 25541 - 25548. [Abstract] [Full Text] [PDF]
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T. A. Ranalli, M. S. DeMott, and R. A. Bambara Mechanism Underlying Replication Protein A Stimulation of DNA Ligase I J. Biol. Chem., January 11, 2002; 277(3): 1719 - 1727. [Abstract] [Full Text] [PDF]
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