(Received for publication, October 11, 1995; and in revised form, January 25, 1996)
From the
Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3` to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3`-flanking nucleotides.
The base excision repair (BER) ()pathway involves the
action of specific DNA glycosylases that catalyze excision of the base,
leaving noncoding apurinic/apyrimidinic (AP) sites in DNA. These
lesions are further processed mainly by the abundant 5`-acting AP
endonucleases, which cleave the DNA backbone generating a 5`-terminal
deoxyribose-phosphate moiety. In Escherichia coli the
5`-nucleotidyl hydrolases, exonuclease III and endonuclease IV,
constitute more than 90% of the total cellular AP endonuclease
activity(1) . In human cells the major 5` AP endonuclease is
termed HAP1 or APE protein (2, 3) and is the human
homologue of the E. coli repair protein, exonuclease III. In E. coli as well as in mammalian cells, the BER excision step
seems to involve only the deoxyribose-phosphate residue with generation
of a single nucleotide gap(4) . DNA polymerase I and DNA
polymerase
have been suggested as the major activities
responsible for gap filling in E. coli and mammalian cells,
respectively(4, 18) .
Recent data obtained both in
prokaryotic and eukaryotic cells raise the question of the existence of
an alternative BER pathway. Two activities have been detected in E.
coli extracts that are able to excise free deoxyribose-phosphate
from DNA: RecJ and MutM gene products. Nevertheless, E. coli fpg/recJ double mutants retain the ability to
repair AP sites, suggesting the existence of backup
systems(5) . In a previous study (6) we have shown that
circular plasmid molecules containing multiple AP sites are efficiently
repaired by Chinese hamster cell extracts in an in vitro repair assay with an estimated repair patch of 6.6 nucleotides for
a single AP site, suggesting that tracts longer than one nucleotide are
synthesized at the lesion site. The analysis of repair of AP sites in a
reconstituted repair system of Xenopus laevis oocytes has
provided very convincing evidence that AP sites are repaired not only
via a DNA polymerase -dependent system but also by a
PCNA-dependent system(7) . Therefore, DNA polymerase
or
seems to function in repair of this ubiquitous form of DNA
damage. Accordingly, evidence has been recently presented that DNA
polymerase
is required for base excision repair of DNA
methylation damage in Saccharomyces cerevisiae(8) .
To investigate whether mammalian cells repair abasic sites via alternative repair pathways a circular DNA duplex molecule was constructed with a single AP site in a defined sequence and the repair patch at the lesion was measured in both hamster and human cell-free repair assays.
We provide the first evidence that, in addition to the single nucleotide insertion pathway, a PCNA-dependent longer patch repair system is active in mammalian cells.
Figure 1: Scheme of the circular duplex DNA molecules used as substrates. pGEM-T and pGEM-U plasmids were obtained by priming single-stranded (+) pGEM-3Zf DNA with the indicated oligonucleotides and performing in vitro DNA synthesis. The single AP site-containing plasmid, pGEM-X, was obtained by incubation of pGEM-U with uracil-DNA-glycosylase. The position of the AP site and the restriction sites utilized for the mapping of the repair patch are indicated.
Figure 2: Characterization of the single AP site-containing substrate. A: lane 1, construct containing a single uracil residue, pGEM-U; lane 2, after incubation with uracil-DNA-glycosylase; lane 3, after incubation with endonuclease III; lane 4, after incubation with uracil-DNA-glycosylase followed by incubation with endonuclease III. B: lane 1, construct containing the control oligonucleotide, pGEM-T; lane 2, after digestion with BamHI (20 units); lane 3, after digestion with AccI (20 units); lane 4, after incubation with uracil-DNA-glycosylase; lane 5, after incubation with endonuclease III. C: lane 6, construct containing a single AP site, pGEM-X; lane 7, after digestion with BamHI (20 units); lane 8, after digestion with AccI (20 units); lane 9, after incubation with endonuclease III.
Figure 3:
Repair of a single AP site by CHO cell
extracts. Autoradiograph of a nondenaturing polyacrylamide gel. Repair
replication was performed in the presence of
[P]dATP (lanes 1, 3, 5, and 7)
or [
P]dTTP (lanes 2, 4, 6, and 8). Left, pGEM-T (lanes 1 and 2) or
pGEM-X (lanes 3 and 4) DNAs were digested with XbaI and HincII to release the 8-bp fragment (A) containing the AP site and nucleotide residues 5` to it. (Right) pGEM-T (lanes 5 and 6) or pGEM-X (lanes 7 and 8) were digested with SalI and PstI to release the 10-bp fragment (B) containing the
AP site and nucleotide residues 3` to it.
The extent of DNA repair replication around the AP site was further characterized by a panel of restriction enzyme digestions as shown in Fig. 4. In all the following experiments only the incorporation of labeled dTMP residues was evaluated. No incorporation was detected in the XbaI-SalI (A) and SmaI-XbaI (D) fragments extending 5` to the lesion, but excluding the AP site. These data together with the lack of dAMP incorporation observed in the XbaI-HincII fragment (see Fig. 3, lane 3), indicate that the incorporation found in the XbaI-AccI fragment (B), originally containing the AP site and the residues 5` to it, is exclusively due to the replacement of the AP site by a single nucleotide. Incorporation was also detected in the HincII-PstI fragment (C), which contains residues 3` to the AP site but not the AP site residue itself. No incorporation was found in the more distal 3` PstI-HindIII fragment (E).
Figure 4:
Characterization of the repair patch at
the AP site. Autoradiograph of a nondenaturing polyacrylamide gel.
Repair replication was performed in the presence of
[P]dTTP. pGEM-X (lanes 1-5) or
pGEM-T (lane 6-10) DNAs were digested with: XbaI and SalI to release the 6-bp fragment (A) containing nucleotide residues flanking 5` the AP site (lanes 1 and 6); XbaI and AccI to
release the 7-bp fragment (B) containing the AP site and
nucleotide residues 5` to it (lanes 2 and 7); HincII and PstI to release the 8-bp fragment (C) containing nucleotide residues flanking 3` to the AP site (lanes 3 and 8); SmaI and XbaI to
release the 9-bp fragment (D) containing nucleotide residues
5` to the AP site (lanes 4 and 9); PstI and HindIII to release the 8-bp fragment (E) containing
nucleotide residues 3` to the AP site (lanes 5 and 10).
These data confirm the occurrence of a repair patch extending 3` to the AP site. Since the incorporation of radiolabeled dTMP was detected at the Thy closest to the AP site (7 nucleotides 3` to the lesion), but not at the Thy located 15 nucleotides 3` to the AP site, the patch of resynthesis could be mapped to a tract of 7-14 nucleotides.
Figure 5: Inhibition of UV-induced DNA excision repair by anti-PCNA polyclonal antibodies. DNA isolated from repair reaction mixtures was visualized by ethidium bromide staining (left panel) or by autoradiography (right panel). The position in the gel of unirradiated pBR322 plasmid DNA (4.3 kb) is indicated by (-UV) and of irradiated pAT153 (3.7 kb) by (+UV). Lanes 1 and 2, repair synthesis without antibody; lane 3-6, after preincubation of cell extracts with anti-PCNA antibody, 0.15 µg (lane 3), 1.5 µg (lane 4), 3.7 µg (lane 5), and 4.6 µg (lane 6).
The role of PCNA in the AP site-induced repair synthesis was investigated. CHO cell extracts were preincubated with 3009 anti-PCNA antibody prior to repair reactions on AP site-containing plasmid DNA. As shown in Fig. 6, the repair synthesis 3` to the AP site (fragment B) was strongly inhibited by anti-PCNA antibody (compare lane 5 with lanes 6 and 7). In contrast, the 1-nucleotide insertion pathway (fragment A) was not inhibited under these conditions (lanes 1-3). The slight increase in repair synthesis observed in the presence of the antibody (compare lane 1 with lanes 2 and 3) indicates that some competition might exist between the two pathways.
Figure 6:
Inhibition of the long patch BER pathway
at the AP site by anti-PCNA polyclonal antibodies. Autoradiograph of a
nondenaturing polyacrylamide gel is shown. Repair replication was
performed in the presence of [P]dTTP. Left, pGEM-X (lanes 1-3) or pGEM-T (lane
4) DNAs were digested with XbaI and AccI to
release the 7-bp fragment (A) containing the AP site and
nucleotide residues 5` to it. Lanes 1 and 4, repair
synthesis without antibody; lanes 2 and 3, after
preincubation of cell extracts with anti-PCNA antibody, 1.5 µg (lane 2) and 3.7 µg (lane 3). Right,
pGEM-X (lanes 5-7) or pGEM-T (lane 8) DNAs were
digested with HincII and PstI to release the 8-bp
fragment (B) containing nucleotide residues 3` to the AP site. Lanes 5 and 8, repair synthesis without antibody; lanes 6 and 7, after preincubation of cell extracts
with anti-PCNA antibody, 1.5 µg (lane 6) and 3.7 µg (lane 7).
These data demonstrate that two
repair pathways are involved in AP site repair in mammalian cells. The
single nucleotide gap filling is PCNA-independent, while the
resynthesis of longer patches extending 3` to the AP site is
PCNA-dependent. Therefore besides DNA polymerase
(4, 18) , PCNA-dependent polymerase(s) (DNA
polymerase
and/or
) might be involved in AP site repair.
Figure 7:
Repair of a single AP site by HeLa cell
extracts. Autoradiograph of a nondenaturing polyacrylamide gel is
shown. Repair replication was performed in the presence of
[P]dTTP and/or [
P]dCTP as
indicated. pGEM-X DNA was digested with FokI (lane 1)
followed by digestion with: SalI and PstI to release
the 10-bp fragment (A) containing the AP site and nucleotide
residues 3` to it (lanes 2, 4, and 6) or HincII and PstI to releases the 8-bp fragment (B) containing nucleotide residues 3` to the AP site (lanes 3, 5, and 7).
The production of repair patches longer than one nucleotide was therefore confirmed in human cell extracts. The higher level of incorporation detected in fragment A, as compared with fragment B, is in agreement with the hypothesis that in mammalian cell extracts two alternative repair pathways for AP sites repair are present.
The BER pathway is a major cellular defense against both spontaneous and chemical/radiation-induced DNA damage. AP sites are generated as a central intermediate in this repair process.
The biological consequences of unrepaired AP sites are numerous. AP sites can block progression of RNA and DNA polymerases, resulting in impairment of gene transcription and DNA replication. If polymerases succeed in bypassing these lesions during transcription or replication, the effects may be even more deleterious for genome stability. Because AP sites are non-instructional lesions to polymerases, there is a high probability that a ``wrong'' base will be inserted opposite an AP site leading to gene mutations (13, 14) or to mutated transcripts(15) . Studies on yeast mutants defective in the major AP endonuclease, APN1, have shown that a failure to rapidly repair AP sites leads to a strong increase in spontaneous mutation frequency(16) . This is presumably due to AP sites arising from the action of endogenous DNA damaging agents such as oxygen radicals or alkylating agents. Similarly, HeLa cell transfectants expressing HAP1 antisense RNA and unable to efficiently repair AP sites are hypersensitive to killing by a range of DNA-damaging chemicals and radiation, including oxygen radical-generating agents and alkylating agents(17) . Thus, the repair of AP sites is essential for suppression of mutations and, therefore, vital for genome stability. It is not surprising if backup systems are employed for the repair of these very common and potentially harmful DNA lesions.
In this study we have provided clear evidence that natural AP sites are repaired by two distinct pathways by mammalian cell extracts. Besides the repair route which involves a single nucleotide gap filling reaction at the abasic site, a new pathway was identified that is PCNA-dependent and involves the replacement of a short oligonucleotide containing the AP site and 6-13 nucleotides 3` to it. Similarly, a PCNA-dependent system for repair of AP sites has been previously identified in X. laevis oocytes(7) .
How much is the relative contribution of the two pathways to the repair of abasic sites still remains an open question. The generation of a single nucleotide repair patch seems to be the favored route for AP site repair in mammalian cell extracts, although the PCNA-dependent pathway is undoubtedly active on natural AP sites. The short patch BER pathway has been shown to be specific for AP site repair, while the long patch BER is also able to repair AP site analogues like the tetrahydrofuran residues(7) . It might well be that in vivo after cell damage the type and rate of AP site production determines whether the long patch BER pathway enters into action as backup system.
In a
recent study aimed at identifying the DNA polymerase(s) requirement for
BER in mammalian nuclear extract(18) , only DNA polymerase
was shown to be required for the gap filling step following
uracil excision. However, it is important to notice that in this study
a 51-bp synthetic oligonucleotide, e.g. a linear
double-stranded molecule, containing a single uracil was used as
substrate. Clear evidence has been provided recently that a stable
RF-C
PCNA complex can be assembled on circular but not on linear
DNA (19) . It has been proposed that RF-C, after binding to
DNA, might load PCNA onto circular duplex DNA and then act as a clamp
to ensure the correct loading of the replicative polymerase at the
synthesis site. The mechanism of assembly of polymerase
or
auxiliary proteins on DNA would then be compromised on a linear
substrate containing the AP site, thus favoring the PCNA-independent,
polymerase
-mediated, repair pathway.
The repair of an AP site
via 1-nucleotide replacement involves the generation of a single
nucleotide gap as a reaction intermediate. DNA polymerase is able
to catalyze both the excision of the 5`-terminal deoxyribose-phosphate
at the incised AP site (20) and DNA synthesis to fill the gap.
The repair of an AP site via the PCNA-dependent pathway might imply the
formation of a flap structure intermediate. A DNA flap is a bifurcated
structure composed of a double-stranded DNA and a displaced
single-strand. It will be of interest to investigate whether, in the
PCNA-dependent pathway, polymerase
/
and 5`-3`-exonuclease of
FEN-1/DNase IV (21, 22) may function together to
replace the damaged bases by nick translation.
In conclusion, these
observations lead to a redefinition of the model of BER in mammalian
cells. As proposed by Dianov and Lindahl (23) for E. coli cells, this excision process is likely to be a branched pathway in
which, besides the very short patch pathway likely mediated by DNA
polymerase , another system enters into the scenario. This repair
system results in the replacement of several nucleotides by a
PCNA-dependent polymerase, DNA polymerase
or
.