From the Laboratory of Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709, the § Department of Human Biological Chemistry and
Genetics, University of Texas Medical Branch, Galveston, Texas 77555, and the ¶ Institute of Biotechnology, Center for Molecular
Medicine, University of Texas Health Science Center,
San Antonio, Texas 78245
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ABSTRACT |
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Base excision repair (BER) is one of the cellular
defense mechanisms repairing damage to nucleoside 5'-monophosphate
residues in genomic DNA. This repair pathway is initiated by
spontaneous or enzymatic N-glycosidic bond cleavage
creating an abasic or apurinic-apyrimidinic (AP) site in
double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide
phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities
complete repair of the AP site. In mammalian cell nuclear extract, BER
can be mediated by a macromolecular complex containing DNA polymerase
(
-pol) and DNA ligase I. These two enzymes are capable of
contributing the latter three of the four BER enzymatic activities. In
the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP
endonuclease,
-pol, and DNA ligase I. Examination of the individual
enzymatic steps in BER allowed us to identify an ordered reaction
pathway: subsequent to 5' "nicking" of the AP site-containing DNA
strand by AP endonuclease,
-pol performs DNA synthesis
prior to removal of the 5'-dRP moiety in the gap. Removal
of the dRP flap is strictly required for DNA ligase I to seal the
resulting nick. Additionally, the catalytic rate of the reconstituted
BER system and the individual enzymatic activities was measured. The
reconstituted BER system performs repair of AP site DNA at a rate that
is slower than the respective rates of AP endonuclease, DNA synthesis,
and ligation, suggesting that these steps are not rate-determining in
the overall reconstituted BER system. Instead, the rate-limiting step
in the reconstituted system was found to be removal of dRP
(i.e. dRP lyase), catalyzed by the amino-terminal domain of
-pol. This work is the first to measure the rate of BER in an
in vitro reaction. The potential significance of the
dRP-containing intermediate in the regulation of BER is discussed.
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INTRODUCTION |
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Base excision repair (BER)1 pathways are employed to repair damaged or modified bases in DNA. Because similar BER pathways are found in prokaryotic and eukaryotic cells, the extensive knowledge about prokaryotic BER has facilitated studies of this repair mechanism in mammalian cells. BER has been examined in vitro with crude extracts from Escherichia coli, Saccharomyces cerevisiae, Xenopus laevis oocyte, bovine testis, and various mammalian cells (1-5) and reconstituted using purified proteins from both prokaryotes and eukaryotes (1, 5-8).
Mammalian cells can repair abasic sites, an intermediate of BER, using
at least two distinct pathways: one involving single nucleotide gap
filling by DNA polymerase ("simple" BER) and an "alternate"
pathway that involves proliferating cell nuclear antigen (PCNA). In
this latter pathway gap-filling DNA synthesis appears to be catalyzed
by DNA polymerase
or
and results in a repair patch of 2-6
nucleotides (9). In addition, Klungland and Lindahl (10) have described
a BER pathway that repairs reduced AP sites. This pathway also
generates a repair patch 2-6 nucleotides in length, but in this case
gap-filling DNA synthesis could be performed by DNA polymerase
(
-pol) or
. Like the pathway described above (9), this BER
pathway was stimulated by PCNA (10).
A working model for the simple BER pathway is outlined as follows (for
review see Refs. 11 and 12). The glycosidic bond linking the damaged
base and deoxyribose is cleaved either spontaneously or by a DNA
glycosylase activity removing the inappropriate base to generate an
abasic or AP site in double-stranded DNA. The phosphodiester backbone
of the AP site is cleaved 5' to the sugar moiety by AP endonuclease,
leaving a 3'-hydroxyl group and a deoxyribose phosphate (dRP) group at
the 5' terminus. Excision of the deoxyribose phosphate group is
catalyzed by 2-deoxyribose-5-phosphate lyase, an activity that is
intrinsic to the amino-terminal 8-kDa domain of -pol. The
-pol
dRP lyase activity functions via
-elimination (13) and produces a
single-nucleotide gap with a 3'-hydroxyl and 5'-phosphate at the gap
margins. DNA polymerase
then fills the single-nucleotide gap, and a
DNA ligase seals the resulting nick.
The identity of the DNA ligase that completes the simple BER pathway in
mammalian cells is unresolved. There is genetic and biochemical
evidence implicating the products of both the LIG1 and
LIG3 genes in BER. Cell lines deficient in either DNA ligase I or DNA ligase III activity are hypersensitive to DNA alkylating agents (14, 15), and extracts from these cell lines are defective in
BER (16, 17). Furthermore, protein-protein interactions between -pol
and XRCC1, the protein partner of DNA ligase III, and between
-pol
and DNA ligase I have been characterized (7, 18, 19). Recently, we
described the partial purification of a BER-proficient multiprotein
complex from bovine testis nuclear extracts (19). DNA polymerase
and DNA ligase I were identified as components of this complex, but no
other ligases were present. Further studies have determined the
stoichiometry and thermodynamic properties of this interaction and
revealed that stable complex formation between DNA ligase I and
-pol
is mediated through the noncatalytic amino-terminal domain of DNA
ligase I and the 8-kDa amino-terminal domain of
-pol (20). Together,
these results support the notion that a complex of
-pol and DNA
ligase I catalyzes the latter steps of simple BER.
To define the influence of this and other putative protein-protein
interactions on catalytic activities of enzymes that participate in
simple BER, we reconstituted BER of a DNA substrate containing an AP
site with three purified human enzymes: AP endonuclease, DNA polymerase
, and DNA ligase I. By characterizing isolated, individual reactions
within the BER pathway, we determined the rate-limiting step. Because
the overall repair of the AP site occurred at a rate similar to that of
dRP removal (dRP lyase step), we suggest that
-pol dRP lyase
activity could determine the choice between the simple and alternate
BER pathways.
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EXPERIMENTAL PROCEDURES |
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Materials--
[-32P]dCTP and
[
-32P]ddATP (specific activity, 6.6 × 106 dpm/pmol) were from Amersham Pharmacia Biotech. High
performance liquid chromatography-purified synthetic 51-base
oligodeoxyribonucleotides that when annealed created a G-U base
pair at position 22 were obtained from Oligos Etc, Inc. (Wilsonville,
OR):
5'-GCTTGCATGCCTGCAGGTCGAUTCTAGAGGATCCCCGGGTACCGAGCTCGA-3' and
3'-CGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCTCGAGCT-5'.
Annealing-- Lyophilized oligodeoxyribonucleotides were resuspended in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the concentrations were determined from their UV absorbance at 260 nm. Complimentary oligodeoxyribonucleotide or template primers were annealed by heating a solution of 10 µM template with an equivalent concentration of oligomers and primer to 90 °C for 3 min and incubating the solution for an additional 15 min at 50-60 °C, followed by slow cooling to room temperature.
Recombinant Human Enzymes--
Human recombinant -pol was
overexpressed from plasmid pWL-11 and purified as described (22).
Oligonucleotide site-directed mutagenesis was performed essentially as
described previously (23). Human AP endonuclease was expressed in
E. coli strain BL21/DE3pLysS from pXC53 carrying the
HAP gene and purified as reported (24). An amino-terminal
84-residue deletion mutant of uracil DNA-glycosylase (UDG) that retains
glycosylase activity was overexpressed in E. coli and
purified to apparent homogeneity using the protocol described (25).
Recombinant human DNA ligase I was overexpressed in
baculovirus-infected cells and purified as described (26).
In Vitro Base Excision Repair Assays--
The simple base
excision repair pathway was reconstituted using the recombinant human
enzymes under the following conditions. The reaction mixture (10 µl)
contained 50 mM Hepes, pH 7.5, 2 mM
dithiothreitol, 0.2 mM EDTA, 100 µg/ml bovine serum
albumin, 10% glycerol, 4 mM ATP, 1 µM 51-bp
substrate with a uracil at position 22, and 0.3 µM
[-32P]dCTP. The enzyme mixture was assembled by mixing
10 nM each UDG, AP endonuclease, and
-pol with 100 nM DNA ligase I at 37 °C for 5 min. Bovine serum albumin
replaced DNA ligase I or other constituent enzymes in the reactions
that were assembled in the absence of an individual enzyme. The
assembled enzyme mixture was incubated with DNA substrate at 37 °C
for 30 min. To follow the reaction time course, the assembled reaction
was incubated in the absence of MgCl2 for 5 min at
37 °C. During the incubation period, uracil is removed by UDG,
creating an AP site. To initiate further repair, 5 mM
MgCl2 (essential for AP endonuclease and
-pol) was
added, and the reaction mixture was incubated at 37 °C. After
various periods, aliquots were removed, and the reaction was terminated
by adding an equal volume of gel loading buffer (40 mM
EDTA, 80% formamide, 0.02% bromphenol blue, and 0.02% xylene cyanol). After 2 min at 95 °C, the reaction products were separated by electrophoresis in a 15% polyacrylamide gel containing 7 M urea in 89 mM Tris-HCl, 89 mM
boric acid, and 2 mM EDTA, pH 8.8. The gel was dried, and
the reaction products were visualized by autoradiography.
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RESULTS |
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Reconstitution of Uracil-initiated Base Excision Repair Using
Purified Human Proteins--
The two substrates in our in
vitro BER system were [-32P]dCTP and a 51-bp
duplex DNA with dUMP at position 22 in one strand (Fig.
1A). The labeled in
vitro BER DNA products are the 51-residue molecule with
[32P]dCMP at position 22 and/or the unligated 22-residue
intermediate. In the experiment shown in Fig. 1B, the BER
system was reconstituted with four purified human enzymes (UDG, AP
endonuclease,
-pol, and DNA ligase I). The reaction mixture was
incubated for 30 min, after which time the 51-residue product and a
very small amount of the unligated 22-residue intermediate accumulated
(lane 1). The ratio of 51-mer to 22-mer observed here was
similar to that observed earlier with bovine tissue and mouse cell line
nuclear extracts (5, 27). The enzyme and substrate requirements for in vitro BER were further investigated. UDG, AP
endonuclease,
-pol, and DNA were each required for radiolabeled
product formation (Fig. 1B, lanes 2-4 and
7). In the absence of DNA ligase I, accumulation of the
unligated 22-residue intermediate was observed (lane 5). With all the enzymes present, the incorporation of
[32P]dCMP into BER products was examined as a function of
time (Fig. 1C). The 22-mer intermediate accumulated modestly
before being ligated to the downstream oligomer.
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Conditions for Kinetic Studies of Base Excision Repair in
Vitro--
To identify potential regulatory steps in BER, we compared
time courses of the reconstituted reaction and the individual steps in
the reaction pathway at 37 °C. These kinetic studies were performed at the enzyme concentrations described in Fig. 1 (10 nM
each UDG, AP endonuclease, and -pol and 100 nM DNA
ligase I) using saturating substrate concentrations (1 µMDNA, 10 µMdCTP, 4 mM ATP) to
allow multiple catalytic turnovers. The decision to use a higher
concentration of DNA ligase I than the other enzymes was based on
preliminary results indicating that the unligated intermediate was
accumulating during BER reactions performed using 10 nM DNA
ligase I (data not shown). Because the persistence of this intermediate
obscured study of enzymatic steps preceding the ligase step in this
pathway (see below) and did not reflect results obtained with tissue
nuclear extract (5), the higher DNA ligase I concentration was used throughout this work to facilitate study of the other steps. The time
course of the reconstituted BER reaction revealed a slight lag phase
preceding a linear steady-state rate (Fig.
2A). Extrapolation of the
linear phase to the abscissa suggested a lag of approximately 5 s.
These results are consistent with a model in which the concentration of
an intermediate initially accumulates in the reaction mixture. After
this lag, the overall base excision reaction proceeded at a velocity of
0.6 nM s
1 (Fig. 2A). The fact that
there was a lag and that the rate of the overall reaction after the lag
was relatively slow suggested that an intermediate accumulates and
limits the overall reaction.
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Identification of the Rate-determining Steps for AP Site BER in
Vitro--
To delineate which BER activities were rate-determining for
product formation, we compared the rates of the various enzymatic activities (Table I). Note that the
velocity measured for the overall BER reaction was 0.6 nM
s1. The first activity measured was the rate of DNA
synthesis. With a single-nucleotide gapped DNA substrate, we found that
-pol formed the 22-residue product at a reaction velocity of 4.5 nM s
1 (Fig. 2B). Addition of DNA
ligase I had a slight stimulatory effect on this DNA synthesis reaction
rate (data not shown). Thus, the rate of the
-pol-mediated
gap-filling reaction on a gapped DNA substrate was faster than the rate
of the overall BER reaction and was not rate-limiting. The presence of
a dRP group at the 5' margin of the gap did not affect this rate (see
Fig. 3A).
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Sequence of Steps in the Reconstituted BER System--
The
sequence of the steps following incision by AP endonuclease was
investigated using both [-32P]dCTP and
3'-32P-labeled DNA to simultaneously measure gap filling
and dRP lyase activity, respectively, in the same reaction mixture. dRP
lyase activity was measured by a difference for the 3' end labeled
substrate and the product strands (Fig.
4, bands 2 and 3,
respectively). The 29-residue product migrates slightly faster than the
29-residue substrate. The product of DNA synthesis is a 22-residue DNA
strand that is well separated from the 3' end-labeled 29-residue
molecules. Gap-filling DNA synthesis was almost complete after 1 min
and was not influenced by the presence of the dRP flap (Fig. 4,
lanes 4 and 13). This provides further evidence
that the dRP group in the gap does not inhibit the DNA synthesis step.
Interestingly, the dRP lyase activity was slightly stimulated
(approximately 2-fold) by the presence of DNA synthesis. However, the
rate of dRP lyase activity in the presence of DNA synthesis remained
slower than the rate of DNA synthesis.
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DISCUSSION |
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A wide variety of exogenous and endogenous chemicals damage DNA bases, initiating their removal by the numerous damage-specific DNA glycosylases present in cells (for review, see Ref. 29). Spontaneous cleavage of the N-glycosidic bond linking both undamaged and damaged nitrogenous bases to the deoxyribose sugar moiety generates additional abasic sites, estimated to total between 2,000 and 10,000 per day per human cell (30). These damaged sites must be repaired in a timely manner, because such noncoding lesions represent gaps in the templates used by polymerases, increasing the likelihood of mutation and aberrant RNA transcripts (31-33). The base excision repair systems in the cell are believed to constitute the primary mechanism for the repair of this form of DNA damage.
This work demonstrates that base excision repair of an abasic site can
be reconstituted in vitro using three human enzymes: AP
endonuclease, DNA polymerase , and DNA ligase I. This is the same
repair system that was studied by Nicholl et al. (8) and is
similar to the pathway described by Kubota et al. (7)
containing AP endonuclease and
-pol but that utilizes a DNA ligase
III-XRCC1 complex in place of DNA ligase I. The possibility that the
DNA ligase III-XRCC1 complex can take the place of DNA ligase I in the
simple BER pathway was not directly examined in this work, so a role
for DNA ligase III in BER is not excluded here. However, a
BER-proficient complex containing
-pol and DNA ligase I, but not DNA
ligase III, has been partially purified from bovine testis nuclear
extract (19). Based on genetic and biochemical analysis of DNA
ligase-deficient mammalian cell lines (14-17), it is possible that DNA
ligase I and DNA ligase III-XRCC1 participate in distinct BER pathways
whose in vivo substrate specificity remains to be elucidated. The simple BER pathway characterized in this study is
distinct from the PCNA-dependent alternate BER pathways,
components of which also play a role in semiconservative DNA
replication and nucleotide excision repair (9, 34, 35).
Our interest in base excision repair stems from studies of mammalian
-pol, which had been proposed to be a DNA polymerase active in
repairing short (1-6 nucleotide) gaps in DNA (5). The identification
of interactions between
-pol and AP endonuclease (36) and between
-pol and DNA ligase I (19, 20), all of which are components of a
BER-proficient complex, strongly supports the notion that the latter
steps of simple BER are catalyzed by the sequential actions of these
enzymes. Experiments by various groups (9, 27, 37) have shown that
simple base excision repair is the predominant type of base excision
repair used by human cells and mouse fibroblast cells; therefore, it
appears likely that the enzymes and reactions studied here constitute the predominant base excision repair pathway operating in human cells.
Importantly, this work measured the catalytic rate of the overall
reconstituted BER reaction as well as individual steps, allowing
identification of the dRP lyase step as the activity likely to be
regulating this pathway. This is the first measurement of the rate of
mammalian base excision repair in an in vitro reaction and,
to our knowledge, the first rate determination of any mammalian DNA
repair system. Within the reconstituted BER system, the dRP lyase
activity was found to be the rate-determining step, because the
velocity of this step (0.75 nM s1) was
similar to the velocity measured for the overall BER reaction (Table
I). The rate of DNA ligase I (4 nM s
1) would
be expected to be partially rate-limiting at lower DNA ligase I
concentrations (e.g. 10 nM). DNA synthesis was
rapid (velocity = 4.5 nM s
1) and not
rate-limiting. An additional finding was that the AP endonuclease
product (dRP-containing intermediate) did not limit DNA synthesis.
Thus, gap filling was not disrupted when the dRP moiety was still bound
to the 5'-phosphate in the gap (see Figs. 3 and 4). Instead, the
presence of a dRP group in the gap was found to inhibit DNA ligase I
activity (see Fig. 4). After removal of the dRP, the rate of BER was
similar to the rate of DNA synthesis and ligation (see Fig. 3). DNA
ligase I was unable to seal the nick while the dRP flap was present,
presumably because the flap interfered with ligation or DNA ligase
binding. Alternatively, binding by
-pol at the gap may prohibit DNA
ligase I from binding to the 5'-phosphate. Although it is known that
the 8-kDa domain of
-pol possesses the dRP lyase active site and
interacts with DNA ligase I (20, 28, 38), the precise molecular
mechanism of this interaction has not been elucidated. Noting that the
dRP lyase step is rate-determining in the BER system and that the dRP
group must be removed prior to ligation, it is clear that dRP removal
plays a significant functional role in the regulation of base excision
repair. These observations also allow us to propose the following order
for the AP site base excision repair enzymatic activities: AP
endonuclease, DNA synthesis, dRP lyase activity, and then ligation
(Fig. 5).
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The identification of the dRP lyase step as a rate-determining step in
simple base excision repair now permits a closer examination of the
pathways capable of repairing AP sites in DNA. Data obtained from
prokaryotic and eukaryotic systems (9, 10, 39-41) suggest that at
least two classes of base excision repair pathway may operate in cells,
including human cells: the simple, -pol-mediated pathway described
here, as well as alternate, PCNA-dependent pathways that
can utilize DNA polymerase
,
, and/or
. It seems plausible that the choice of pathway would be linked to the status of the dRP
group. Should the dRP be processed quickly by the dRP lyase activity of
-pol, the simple BER mechanism would likely complete the repair of
the gap. If the dRP group were to persist in the DNA, however, it seems
possible that the components of an alternate base excision repair
pathway might bind to the dRP flap (with or without
-pol bound at
the site) and complete the repair event. Thus, the status of the dRP
group might function as a "switch" between the simple and alternate
repair pathways. Although both pathways are known to occur in
competition with each other in human cells (9), characterization of the
signal that initiates each alternate BER pathway on gapped DNA has not
been elucidated.
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FOOTNOTES |
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* 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: Lab. of Structural
Biology, NIEHS, 111 Alexander Dr., Bldg. 101, Rm. B246, Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax: 919-541-2260; E-mail:
wilson5{at}niehs.nih.gov.
The abbreviations used are:
BER, base excision
repair; AP, apurinic/apyrimidinic; dRP, deoxyribose phosphate; -pol, DNA polymerase
; UDG, uracil DNA-glycosylase; PCNA, proliferating
cell nuclear antigen; XRCC1, x-ray repair cross-complementing protein
1; bp, base pair(s).
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REFERENCES |
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