The Type of DNA Glycosylase Determines the Base Excision
Repair Pathway in Mammalian Cells*
Paola
Fortini
,
Eleonora
Parlanti
,
Olga M.
Sidorkina§,
Jacques
Laval§, and
Eugenia
Dogliotti
¶
From the
Laboratory of Comparative Toxicology and
Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena
299, 00161 Rome, Italy and § Groupe Réparation de
l'ADN, UMR 1772 Centre National de la Recherche Scientifique, Institut
Gustave Roussy, 39, rue Camille Desmoulins,
94805 Villejuif Cedex, France
 |
ABSTRACT |
The base excision repair (BER) of modified
nucleotides is initiated by damage-specific DNA glycosylases. The
repair of the resulting apurinic/apyrimidinic site involves the
replacement of either a single nucleotide (short patch BER) or of
several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair
of the resulting abasic site intermediate to either BER branch. In
mammalian cells hypoxanthine (HX) and
1,N6-ethenoadenine (
A) are both substrates
for the monofunctional 3-methyladenine DNA glycosylase, the ANPG
protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the
bifunctional DNA glycosylase/
-lyase 8-oxoG-DNA gly- cosylase
(OGG1). Circular plasmid molecules containing a single HX,
A, or
8-oxoG were constructed. In vitro repair assays with HeLa
cell extracts revealed that HX and
A are repaired via both short and
long patch BER, whereas 8-oxoG is repaired mainly via the short patch
pathway. The preferential repair of 8-oxoG by short patch BER was
confirmed by the low efficiency of repair of this lesion by DNA
polymerase
-deficient mouse cells as compared with their wild-type
counterpart. These data fit into a model where the intrinsic properties
of the DNA glycosylase that recognizes the lesion selects the branch of
BER that will restore the intact DNA template.
 |
INTRODUCTION |
Various DNA-damaging agents produce modified bases in DNA that are
repaired by the base excision repair
(BER)1 pathway (reviewed in
Ref. 1). A number of DNA glycosylases recognize the damaged bases and
remove them through N-glycosidic bond hydrolysis. There are
two types of DNA-glycosylases: the monofunctional exhibiting only the
glycosylase activity and the bifunctional, which are
glycosylase/
-lyases. In human cells, an example of the first
category is the 3-methyladenine-DNA glycosylase, the ANPG protein.
It catalyzes the excision of a broad variety of modified bases
including N-methylpurines generated by alkylating agents
(reviewed in Ref. 1), deamination products like hypoxanthine (HX) (2),
and 1,N6-ethenoadenine (
A), an adduct
generated by chloroacetaldehyde or products of lipid peroxidation (3,
4). An example of a DNA glycosylase with an associated AP-lyase is
hOGG1, the human homolog of the Escherichia coli Fpg protein
(formamidopyrimidine-DNA glycosylase) (5), which excises the potent premutagenic
lesion 7,8-dihydroxy-8-oxoguanine (8-oxoG) (6-11). The removal of
8-oxoG is followed by DNA strand cleavage by hOGG1 via
-elimination (reviewed in Ref. 1).
This damage-specific initial step, carried out by individual DNA
glycosylases, is followed by the processing of the resulting apurinic/apyrimidinic (AP) site, a mutagenic repair intermediate (reviewed in Ref. 12), presumably by the major mammalian
AP-endonuclease, HAP1/APEX (reviewed in Ref. 13). AP sites are
processed via two alternative pathways: the short patch (1-nucleotide
gap filling) (14-16) and the long patch (2-6 nucleotide resynthesis)
BER (17-19). These two pathways involve some common proteins but also
some specific ones. For example in the short patch pathway, DNA
polymerase (Pol)
is involved in the resynthesis step (20), whereas
PCNA and Pol
/
are implicated in the long patch pathway (21). It therefore becomes important to investigate whether the initial recognition step of the modified base by a specific DNA glycosylase targets the following repair steps to a specific BER branch. In this
study, specific lesions for the two types of DNA-glycosylases, namely
HX and
A for the monofunctional ANPG protein and 8-oxoG for the
bifunctional OGG1 protein were selected, and their respective DNA
repair pathways were analyzed. Circular duplex plasmids containing a
single lesion at a known position of their genome were used as DNA
substrates to distinguish the two BER branches by fine mapping of the
repair synthesis patches. We report that HX and
A are repaired via
the short as well as the long patch BER, wheras the 8-oxoG residues are
preferentially repaired via the short patch pathway. Because Pol
is
involved in the short patch repair, we show, as expected, that Pol
-null mouse cell extracts exhibited a significantly slower repair of
8-oxoG residues as compared with their wild-type couterpart.
 |
EXPERIMENTAL PROCEDURES |
Chemicals, Enzymes, and Cell-free Extracts--
Chemicals were
purchased from Sigma, and molecular biological reagents were from Roche
Molecular Biochemicals or New England Biolabs.
[
-32P]dATP, [
-32P]dCTP,
[
-32P]dTTP, and [
-32P]dGTP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. T4 DNA
polymerase holoenzyme, single-stranded DNA binding protein and T4 DNA
ligase were purchased from Roche Molecular Biochemicals.
For the oligodeoxyribonucleotides containing a single uracil,
HX,
A, or 8-oxoG residue, 5'-GATCCTCTAGAGUCGACCTGCA-3' and 5'-GATCCTCTAGAGTCG(HX)CCTGCAGGCATGCA-3' were synthesized by M-Medical (Florence, Italy), and 5'-GATCCTCTAGAGTCG(
A)CCTGCAGGCATGCA-3' was
synthesized by Genset (Paris, France) and 5'-GATCCTCTAGAGTC(8oxoG) ACCTGCAGGCATGCA-3' was synthesized by Eurogentec (Angers,
France). These oligonucleotides were used to create duplex plasmid
molecules containing a single lesion at a defined position.
The recombinant Nth protein (endonuclease III), ANPG, and Fpg proteins
were purified to homogeneity as described in Refs. 4, 5, and 22.
E. coli uracil-DNA glycosylase, UDG protein, was a gift of
Dr. S. Boiteux (Centre Energie Atomique, Fontenay aux Roses, France).
Purified recombinant rat Pol
was a gift of Dr. J. S. Hoffmann
(CNRS, Institut de Pharmacologie et Biologie Structurale, Toulouse,
France). Whole cell extracts from HeLa cells and wild-type and Pol
-null mouse fibroblasts (a gift of Dr. S. H. Wilson, NIEHS,
Research Triangle Park, NC) were prepared as described previously (18,
23). To separate PCNA from other repair proteins, whole cell extracts
were chromatographed onto a phosphocellulose column as described (24).
Under these experimental conditions, the flow-through fraction (CFI)
contained PCNA, and the bound fraction (CFII) contained all proteins
essential for short patch BER. We have verified that the addition of
PCNA to CFII is required to observe the long patch
BER.2
Preparation of DNA Substrates--
Closed circular DNA
containing a single lesion was produced as described previously (18) by
priming single-stranded (+) pGEM-3Zf DNA (Promega) with the
oligonucleotide containing the modified base of interest. It was
further incubated with T4 DNA polymerase holoenzyme, single-stranded
DNA binding protein, dNTPs, and T4 DNA ligase. Closed circular DNA
duplex molecules were purified by cesium chloride equilibrium
centrifugation. The plasmid DNA containing a single uracil residue was
digested with UDG to produce a single abasic site. The oligonucleotide
5'-GATCCTCTAGAGTCGACCTGCA-3' was used to prepare the control plasmid.
In Vitro Repair Assays--
Repair reactions were carried out
essentially as described in Ref. 18. Briefly, reaction mixtures (50 µl) contained 40 mM Hepes/KOH (pH 7.9), 75 mM
KCl, 5 mM MgCl2, 0.5 mM
dithiothreitol, 20 µM of each dNTP, 2 µCi of
[
-32P]dATP, [
-32P]dCTP,
[
-32P]dTTP, or [
-32P]dGTP as
indicated, 2 mM ATP, 40 mM phosphocreatine, 2.5 µg of creatine phosphokinase (type I, Sigma), 3.4% glycerol, 18 µg
of bovine serum albumin, and 20 µg of human cell extracts. After increasing periods of time at 30 °C, the plasmid DNA was recovered and digested with restriction enzymes as indicated. The digestion products were electrophoresed on a denaturing 15% polyacrylamide gel.
The repair products were visualized by autoradiography and quantified
by electronic autoradiography (Istant Imager, Packard).
 |
RESULTS |
Characterization of the DNA Substrates Containing Different
Lesions--
Single-stranded (+) pGEM-3Zf DNA was primed with 5'-end
32P-labeled oligonucleotide containing the specific
modified base, and DNA was synthesized in vitro using this
primed template. As shown in Fig. 1, in
all cases closed circular molecules (Form I) were obtained with an
efficiency close to 100% (lanes 1, 5,
8, and 11). When the duplex DNA molecules
containing the lesion were digested with the specific monofunctional
DNA-glycosylase, namely the UDG protein in the case of the uracil
residue (lane 2) and ANPG protein in the case of HX
(lane 6) and
A (lane 9) lesions, followed by
Nth protein (that incises DNA at AP sites by a
-elimination mechanism), a complete conversion of Form I to Form II molecules was
observed (lanes 4, 7, and 10). In the
case of the plasmid containing the 8-oxoG residue (lanes 11 and 12), the incubation with the Fpg protein was sufficient
per se to convert supercoiled plasmids to nicked circular
forms because this enzyme is also endowed with an AP-lyase activity
that incises at AP sites by a
-
elimination mechanism (25, 26).
The recombinant plasmid molecules were partially or fully resistant to
cleavage by restriction enzymes (SalI, AccI, and
HincII) whose recognition sequence include the modified base
confirming that a single lesion was precisely inserted at the expected
position of the plasmid genome (data not shown). These data show that
these duplex DNA molecules are homogeneous closed circular molecules
all containing a single modified base.

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Fig. 1.
Characterization of single lesion containing
substrates. Lane 1, construct containing a single
uracil residue (U); lane 2, after incubation with
UDG; lane 3, after incubation with Nth; lane 4,
after incubation with UDG followed by incubation with Nth; lane
5, construct containing a single HX: lane 6, after
incubation with APNG; lane 7, after incubation with APNG
followed by incubation with Nth; lane 8, construct
containing a single A; lane 9, after incubation with
APNG; lane 10, after incubation with APNG followed by
incubation with Nth; lane 11, construct containing a single
8-oxoG; lane 12, after incubation with Fpg.
|
|
HX and
A Are Processed via Both Short and Long Patch
BER--
To distinguish between the short and long patch BER pathways,
the experimental approach involves the use of different labeled dNTPs
in the reaction mixture. The incorporation of the specific radiolabeled
dNTP at the nucleotide position where the lesion was originally located
within the XbaIHindIII restriction fragment (24 nucleotides) marks mainly the occurrence of one-gap filling reactions, whereas the incorporation of dCMP identifies long repair patches (Fig. 2, see scheme at
bottom) (27). Moreover, because PCNA is required for the
long patch but not for the short patch BER (18), the dependence of the
repair reaction on this auxiliary protein is an additional marker to
distinguish between the two pathways.

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Fig. 2.
Repair of HX and A
by HeLa cell extracts. Repair replication was performed for
different periods of time in the presence of
[ -32P]dATP or [ -32P]dCTP as
indicated. The plasmid DNA was then digested with
XbaI-HindIII to release the 24-bp fragment
originally containing the lesion. A, left,
autoradiograph of a denaturing polyacrylamide gel showing repair
synthesis at the HX residue. Lanes 1-3, short patch repair
synthesis as a function of the incubation time; lanes 4-6,
long patch repair synthesis as a function of the incubation time.
B, left, autoradiograph of a denaturing
polyacrylamide gel showing repair synthesis at the A residue.
Lanes 1-3, short patch repair synthesis as a function of
the incubation time; lanes 4-6, long patch repair synthesis
as a function of the incubation time. A and B,
right, the repair products were measured by electronic
autoradiography and relative incorporation, corrected for DNA recovery,
is indicated on the ordinate (Net CPM).
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|
The deamination of adenine residues in DNA generates HX, whereas lipid
peroxidation generates exocyclic adducts and among them
A. Both HX
and
A are repaired, in human cells, by the same gene product, the
ANPG protein. As shown in Fig. 2, HeLa cell extracts were proficient in
the repair of these modified bases via both pathways: replacement of
one nucleotide (Fig. 2, A and B, lanes
1-3) and resynthesis of longer patches (Fig. 2, A and B, lanes 4-6). Besides the radiolabeled 24-mer,
which is a marker of fully repaired DNA, the other radioactively
labeled DNA present on the gel autoradiography is a 5' end-labeled
60-mer internal standard added in all reaction mixtures. This internal
standard is used to correct the repair incorporation values (as
measured by electronic autoradiography; Fig. 2, right
panels) for DNA recovery.
A comparison of the nature and the relative contribution of the two BER
pathways when using plasmids containing either HX or
A with the
repair of preformed AP site showed that all three lesions were repaired
by the same pathways with a comparable efficiency (data not shown),
suggesting that the AP site processing could be the rate-limiting step
in the repair initiated by the ANPG protein.
8-oxoG Is Repaired Preferentially via Short Patch BER--
In
mammalian cells 8-oxoG is excised from oxidatively damaged DNA by the
enzyme OGG1, which excises 8-oxoG residue when paired with cytosine and
then nicks DNA next to the AP site (28). Fig. 3A shows that crude HeLa cell
extracts were able to repair this oxidized guanine in vitro
but mainly via the short patch BER (lanes 1-3). DNA repair
patches longer than 1-2 nucleotides were undetectable on the gel
autoradiography after up to 2 h of repair time (lanes 4-6). The absence of significant incorporation of dCMP into the repair fragment indicates that 8-oxoG is a poor substrate for long
patch BER, at least extending beyond two nucleotides. Moreover, it also
strongly suggests that nucleotide excision repair (NER) is barely or
not involved in the repair of this lesion under our experimental
conditions. This fact is confirmed by the lack of repair synthesis in
the XbaI-HincII digestion fragment (lane
14), which includes 7 nucleotides 5'-flanking the lesion.

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Fig. 3.
Repair of 8-oxoG as compared with a preformed
AP site by HeLa cell extracts. Repair replication was performed
for different periods of time in the presence of
[ -32P]dGTP, [ -32P]dCTP, or
[ -32P]dTTP as indicated. The plasmid DNA was then
digested with XbaI-HindIII to release the 24-bp
fragment originally containing the lesion. A, autoradiograph
of a denaturing polyacrylamide gel. Lanes 1-6, repair
synthesis at the 8-oxoG residue. Lanes 1-3, short patch
repair synthesis as a function of the incubation time; lanes
4-6, long patch repair synthesis as a function of the incubation
time. Lanes 7-12, repair synthesis at the AP site.
Lanes 7-9, short patch repair synthesis as a function of
the incubation time; lanes 10-12, long patch repair
synthesis as a function of the incubation time. Lane 13,
control plasmid. Lane 14, following repair replication, the
plasmid DNA containing 8-oxoG was digested with
XbaI-HincII to release a 8-bp fragment as shown
in the scheme. B, repair efficiency of 8-oxoG as
compared with a preformed AP site. The repair products were measured by
electronic autoradiography and relative incorporation, corrected for
DNA recovery, is indicated on the ordinate (Net
CPM).
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The comparison of the extent of repair of the 8-oxoG lesion
(lanes 1-6) with that of an AP site constructed in the same
circular plasmid (lanes 7-12) showed that the repair
activity toward an AP site was significantly higher than the specific
repair of 8-oxoG residues (Fig. 3B). This result suggests
that in the repair of oxidative DNA damage, the efficiency of the
initial step(s), up to the production of 3' OH primers for repair
synthesis, determines the overall repair activity.
Short Patch BER of 8-oxoG Is PCNA-independent and Involves Mainly
the Replacement of a Single Nucleotide--
PCNA is not only a protein
required for DNA replication but also plays a key role in long patch
BER and NER (reviewed in Ref. 29). A purified HeLa cell extract
fraction, CFII, containing all the components required for BER except
PCNA (data not shown) was used to verify whether the repair of 8-oxoG
was affected by the presence of PCNA. The CFII fraction (Fig.
4, lanes 4-6) was able to
perform the short patch BER of the oxidized base. The addition of PCNA
did not modify the extent of DNA repair in the case of short incubation
times (lanes 7 and 8), whereas an increase of the
full repair product was observed after 2 h of repair (lane 9). This increase is likely to reflect filling in of gaps longer than 1 nucleotide due to strand displacement reactions stimulated by
PCNA. Because the repair reaction is largely independent from the
presence of PCNA, this is an additional argument suggesting that
neither NER nor long patch BER play a significant role in the repair of
this lesion.

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Fig. 4.
Requirement of PCNA for repair synthesis at
8-oxoG by HeLa cell extracts. Repair replication was performed for
different periods of time in the presence of
[ -32P]dGTP. The plasmid DNA was then digested with
XbaI-HindIII to release the 24-bp fragment
originally containing the lesion. Top, autoradiograph of a
denaturing polyacrylamide gel is shown. Lanes 1-3, repair
synthesis by whole cell extracts (WCE) as a function of the
incubation time; lanes 4-6, repair synthesis by the CFII
fraction as a function of the incubation time; lanes 7-9,
repair synthesis by the CFII fraction after addition of PCNA (50 ng) as
a function of the incubation time. Bottom, the repair
products were measured by electronic autoradiography, and relative
incorporation, corrected for DNA recovery, is indicated on the
ordinate (Net CPM).
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A fine mapping of the repair patch was performed using different
radiolabeled dNTPs in the repair reaction. Fig.
5 shows that the large majority of the
nucleotide replacements in the reactions are confined to 1 nucleotide
(lanes 1-3), approximately one-third of the repair events
involve 2 nucleotides (lanes 4-6), and very rarely exceed
this size including 3-7 nucleotides downstream to the lesion
(lane 7). As expected, no repair synthesis was detected in
the undamaged control plasmid (lane 8).

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Fig. 5.
Characterization of the repair patch at
8-oxoG by HeLa cell extracts. After repair replication the plasmid
DNA was digested with XbaI-HindIII to release the
24-bp fragment originally containing the lesion. Top,
autoradiograph of a denaturing polyacrylamide gel. Lanes
1-3, repair synthesis in the presence of
[ -32P]dGTP as a function of the incubation time;
lanes 4-6, repair synthesis in the presence of
[ -32P]dATP as a function of the incubation time;
lane 7, repair synthesis in the presence of
[ -32P]dCTP after 180 min of incubation time;
lane 8, control plasmid. Bottom, the repair
products were measured by electronic autoradiography, and relative
incorporation, corrected for DNA recovery, is indicated on the
ordinate (Net CPM).
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Pol
Is the DNA Polymerase of Election in 8-oxoG
Repair--
Pol
is the major DNA polymerase implicated in the
single-nucleotide gap filling synthesis in BER (27, 30, 31). Therefore, extracts from mouse Pol
-knockout cells and their isogenic wild-type cells (20) were tested for their ability to repair 8-oxoG residues. As
shown in Fig. 6, we observed a striking
difference in the amount of repair when wild-type cell extracts
(lanes 1-4) were compared with Pol
-defective extracts
(lane 5-8). In the absence of Pol
, almost no repair was
detected after 1 h of repair time (lane 6), whereas the
repair process using wild-type cells extracts had by that time reached
a plateau (Fig. 6B). Moreover, when purified rat Pol
(10 ng) was added to the Pol
-null extract (Fig.
7, lanes 7 and 8),
the repair activity was restored to the level measured with wild-type
extracts (lanes 1 and 2). Taken all together these results strongly suggest that Pol
is the polymerase of election for filling in the gap created by the excision of 8-oxoG. Pol
-defective extracts were eventually able to repair the gap after
3 h of incubation (Fig. 6A, lane 8),
suggesting a possible back-up system that is Pol
-independent.

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Fig. 6.
Repair of 8-oxoG by wild-type and Pol
-deleted mouse cell extracts. Repair
replication was performed for different periods of time in the presence
of [ -32P]dGTP. The plasmid DNA was then digested with
XbaI-HindIII to release the 24-bp fragment
originally containing the lesion. A, autoradiograph of a
denaturing polyacrylamide gel. Lanes 1-4, repair synthesis
by wild-type cell extracts as a function of the incubation time;
lanes 5-8, repair synthesis by Pol -null cell extracts
as a function of the incubation time. B, the repair products
were measured by electronic autoradiography, and relative
incorporation, corrected for DNA recovery, is indicated on the
ordinate (Net CPM).
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Fig. 7.
Effect of additional Pol on 8-oxoG repair by wild-type and Pol
-deleted mouse cell extracts. Repair
replication was performed for different periods of time in the presence
of [ -32P]dGTP. The plasmid DNA was then digested with
XbaI-HindIII to release the 24-bp fragment
originally containing the lesion. Top, autoradiograph of a
denaturing polyacrylamide gel. Lanes 1 and 2,
repair synthesis by wild-type cell extracts as a function of the
incubation time; lanes 3 and 4, after addition of
10 ng of purified rat Pol ; lanes 5 and 6,
repair synthesis by Pol -null cell extracts as a function of the
incubation time; lanes 7 and 8, after addition of
10 ng of purified rat Pol . Bottom, the repair products
were measured by electronic autoradiography, and relative
incorporation, corrected for DNA recovery, is indicated on the
ordinate (Net CPM).
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Mouse cell extracts were tested for their ability to perform long patch
BER at 8-oxoG lesions. As shown in Fig.
8, mouse extracts were unable to perform
detectable repair synthesis of three or more nucleotides at this lesion
(lanes 1-3) extending to other species the results reported
above for human cells. This type of long patch repair synthesis was
only detected with Pol
-defective extracts (lanes 4-6),
although the extent of activity was at the threshold of detection
(lane 6).

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Fig. 8.
Long patch repair synthesis at 8-oxoG by
wild-type and Pol -deleted mouse cell
extracts. Repair replication was performed for different periods
of time in the presence of [ -32P]dCTP. The plasmid DNA
was then digested with XbaI-HindIII to release
the 24-bp fragment originally containing the lesion. Autoradiograph of
a denaturing polyacrylamide gel is shown. Lanes 1-3, repair
synthesis by wild-type cell extracts as a function of the incubation
time; Lanes 4-6, repair synthesis by Pol -null cell
extracts as a function of the incubation time. IS, internal
standard.
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 |
DISCUSSION |
In the BER pathway, the initial step is mediated by specific
DNA-glycosylases that excise the modified base and thus generate an AP
site. There are two classes of DNA glycosylases. An example of the
first one is the ANPG protein, the human 3-methyladenine DNA
glycosylase, a homolog of the E. coli Alk A protein, whose only catalytic function is the cleavage of the
C1'-N-glycosydic bond, liberating the modified base and
generating an AP site in DNA. This AP site is believed to be further
processed by the HAP1/APEX protein, an AP endonuclease that generates
on the 5' side a nick having a 3'-hydroxyl group (reviewed in Ref. 13).
An example of the second class of DNA glycosylase is the OGG1 protein,
the homolog of the E. coli Fpg protein, that excises
oxidized purines. Besides its DNA glycosylase activity, this protein is
endowed with a AP lyase activity that nicks DNA at AP site by a
-elimination mechanism (28, 32). It generates on the 3' side a nick
having a 5'-phosphate. Therefore the products generated by these two classes of glycosylases are different. In mammalian cells, two pathways
are involved in AP site repair, the short patch (1-nucleotide gap
filling) and the long patch (2-6 nucleotide resynthesis) BER (reviewed
in Ref. 33). We hypothesized that structural different modified bases,
being recognized by DNA glycosylases having different intrinsic
properties, will drive the repair process to either the short or the
long patch pathway. In this study we showed that, indeed, HX and
A,
excized by the monofunctional ANPG protein, are repaired via both the
short and long patch BER, whereas 8-oxoG residues, excized by the
bifunctional OGG1 protein, are repaired mainly by the short patch pathway.
In the case of HX and
A, under our experimental conditions, the
relative efficiency of the two BER branches was similar to what
previously reported for the repair of preformed AP site, suggesting
that the initial step, i.e. the cleavage by the DNA N-glycosylase, is not rate-limiting. In the case of 8-oxoG, the repair
kinetics was much slower than that observed for the repair of preformed
abasic site, suggesting that in this case the initial step(s),
i.e. the cleavage by the glycosylase/AP lyase and/or the 3'
terminus-removing activity, could drive the efficiency of the process.
In E. coli 8-oxoG residues are repaired by both BER and NER
(34). Our data show that in mammalian cells most of 8-oxoG lesions are
repaired by the short patch BER in a PCNA-independent mechanism. However, our observations do not preclude a small contribution of NER
to repair of oxidative DNA damage (35), which might go undetected in
our repair assay.
To attain the sensitivity required for detection of the repair patches
we used appropriately purified circular duplex substrates containing
modified nucleotides having stable glycosidic bonds located at specific
positions of the plasmid genome. The lesions that are introduced by
exogenous damaging DNA compounds generate substrates that have serious
drawbacks; the specificity is not absolute, and the lesions are
randomly distributed in the molecule. The use of different types of
damaged DNA substrates (single lesion versus randomly
damaged plasmids) might explain some discrepancies between our findings
and those by Jaiswal et al. (36) on the repair pathways at
8-oxoG lesions.
Based on our results we propose that the type of DNA glycosylase that
initiates the BER process determines which branch of BER is selected to
restore the original DNA template. Our findings suggest a model where
the formation of a 5' abasic terminus by the sequential action of a
monofunctional glycosylase, like ANPG, and of a 5'-AP endonuclease,
like HAP1/APE, and its slow processing by a dRPase activity will
determine the long patch repair events that occur in competition with
the predominant one-gap filling reactions. The status of the 5'
terminus would then have a functional role as already suggested by the
finding that the dRP lyase activity of Pol
is rate-determining in a
reconstituted BER system in vitro using AP endonuclease, Pol
, and DNA ligase I (16).
In agreement with this model, the formation of a 3' blocked terminus by
the glycosylase/AP-lyase activity of OGG1 is preferentially followed by
single nucleotide replacement reactions. In this case the determinant
of the repair synthesis step could be the production of 3' OH primers
(likely by the 3' phosphoesterase activity of HAP1) because the 5'
terminus produced by the AP-lyase is ready for the ligation step.
While this manuscript was under revision, two studies were published on
the BER of oxidative DNA damage. Klungland et al. (37), by
reconstituting in vitro the BER of oxidized pyrimidines that
are a substrate for the hNth protein, a bifunctional DNA glycosylase
(reviewed in Ref. 1), showed that this type of BER is strictly a
1-nucleotide replacement pathway. A model similar to that inferred from
our data is proposed implying that the presence of a genuine 5'
nucleotide residue minimizes strand displacement events. The second
paper by Dianov et al. (38) reported that mammalian cell
extracts repair 8-oxoG lesions preferentially via single nucleotide
replacement reactions (75% of the repair events). In this study, in
agreement with the findings reported above, the contribution of NER to
the repair of this lesion was not significant.
The limited efficiency of repair of 8-oxoG, also observed by Dianov
et al. (38), leaves open the question of whether our experimental conditions are optimal for detecting this repair process.
It is not possible to rule out that cofactors that might stimulate, for
example, the affinity of the DNA glycosylase for the target lesion are
lost or not functional in our assay. Moreover, several reports
suggest that the repair of oxidative lesions might be inducible. The
major mammalian AP endonuclease, HAP1/APEX, whose 3' phosphoesterase
activity removes 3' blocking groups generated in DNA by glycosylase/AP
lyase activity, has been shown to be activated specifically by reactive
oxygen species (39). This activation is paralleled by an increased cell
resistance to the cytotoxicity of reactive oxygen species generating
agents. These findings are compatible with the hypothesis that HAP1,
via its phosphoesterase activity, is the rate-limiting step in BER of oxidative lesions.
In previous studies (21, 23) we have shown that Pol
-defective cell
extracts are able to compensate for the lack of Pol
by using Pol
and/or
for repair synthesis following AP site incision. Also in
the case of 8-oxoG repair back-up systems seem to be able to substitute
for Pol
because Pol
-null cells are eventually able to repair
these lesions although at a much slower rate than wild-type cells. The
understanding of the biological relevance of these back-up repair
systems in vivo waits further studies on the genotoxic
effects in Pol
-null cells of various BER-inducing agents including
reactive oxygen species generators.
 |
ACKNOWLEDGEMENT |
We are grateful to L. Gargano for technical assistance.
 |
FOOTNOTES |
*
This work was partially supported by European Community
Grant ENV4-CT97-0505 and by grants from CNRS, the Fondation
Franco-Norvégienne and l'Association pour la Recherche sur le
Cancer (to J. L.), and Associazione Italiana per la Ricerca sul
Cancro (to E. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed.
2
B. Pascucci and E. Dogliotti, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
BER, base excision
repair;
NER, nucleotide excision repair;
AP, apurinic/apyrimidinic;
Pol
,
, and
, DNA polymerase
,
, and
;
HX, hypoxanthine;
A, 1,N6-ethenoadenine;
8-oxoG, 7,8-dihydro-8-oxoguanine;
bp, base pair;
PCNA, proliferating cell
nuclear antigen;
ANPG, alkyl N purine glycosylase.
 |
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