(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
(
-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.
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) ()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) ).
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:CA: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.
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 [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.
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`-P(*) label.
To establish the position of [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).
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 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.
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 -pol-mediated reaction.
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 -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.
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) 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.
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 50 kDa, except for DNA ligase.
Figure 6:
Reconstitution of the repair reaction and
its inhibition by -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.
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 (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.
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 -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.
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 -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.
Next, we
prepared nuclear extract from the S. cerevisiae strain
LP3041-6D (wild type) and the strain JWY355 carrying a -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.
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 (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.
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 -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.
In conclusion, we
have demonstrated by several criteria that the uracil-initiated BER DNA
polymerase activity in our crude extract is -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.
Addendum-DNA
polymerase is able to conduct repair of only natural AP sites in
abasic site repair in X. laevis oocytes(34) .