From the § Laboratory of Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709 and the Sealy Center for Molecular Science,
University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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DNA polymerase (
-pol) cleaves the
sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site
(i.e. AP lyase activity). The same bond is cleaved even if
the AP site has been previously 5'-incised by AP endonuclease,
resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase
activity). We characterized these lyase reactions by steady-state
kinetics with the amino-terminal 8-kDa domain of
-pol and with the
entire 39-kDa polymerase. Steady-state kinetic analyses show that the
Michaelis constants for both the dRP and AP lyase activities of
-pol
are similar. However, kcat is approximately
200-fold lower for the AP lyase activity on an intact AP site than for
an AP endonuclease-preincised site. The 8-kDa domain was also less
efficient with an intact AP site than on a preincised site. The
full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP
from a preincised AP site in the absence of Mg2+, and the
pH profiles of
-pol and 8-kDa domain dRP lyase catalytic efficiency
exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal
5'-phosphate on the dRP lyase activity is consistent with involvement
of a primary amine (Lys72) as the Schiff base nucleophile
during lyase chemistry.
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INTRODUCTION |
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The cellular genome suffers extensive damage from exposure to ultraviolet light and ionizing radiation and also from alkylating agents and reactive oxygen species that accumulate in cells due to environmental stress and natural metabolic processes (1). These DNA lesions occur at a frequency far too high to be compatible with life unless they are repaired to allow faithful function and reproduction of the genome. To remove such damage and restore the integrity of the genome, specific DNA repair pathways have evolved. The major pathway, which protects cells from the deleterious effects of nucleotide base DNA damage, is known as DNA base excision repair (BER).1 BER is initiated by removal of a damaged or inappropriate base residue in DNA by cleavage of the N-glycosidic bond (1, 2). If not repaired, the resulting abasic or apurinic/apyrimidinic (AP) site may result in mutations, altered gene expression, chromosome breakage, apoptosis, and/or cell death.
Both procaryotes and eucaryotes possess class II AP endonucleases that
cleave 5' to an AP site leaving a 3'-hydroxyl and a 5' 2-deoxyribose
5-phosphate (dRP) in the nicked DNA (Fig. 1). In Escherichia
coli, two major AP endonuclease genes, xth and nfo, have been identified encoding exonuclease III and
endonuclease IV, respectively (3, 4). Homologous genes to
xth have been cloned from many sources, including
Drosophila melanogaster and human cells (5-7). The major AP
endonuclease in yeast, APN1, was shown to be an endonuclease IV
homologue (8). These class II AP endonucleases also possess
3'-phosphodiesterase and 3'-phosphatase activities, and some members of
the xth family carry 3' to 5' exonuclease activity (9).
Subsequent to AP endonuclease cleavage in BER, the cleaved AP site must
be processed further (Fig. 1). The enzymatic removal of the
sugar-phosphate residue at the 5' terminus by a dRP lyase mechanism
yields 4-hydroxy-2-pentenal-5-phosphate via -elimination.
Alternatively, hydrolytic cleavage releases dRP. The hydrolytic enzymes
require Mg2+ for activity (10-13).
Another class of AP endonuclease is termed AP lyase. These enzymes
cleave the AP site 3' to the sugar by a -elimination mechanism, leaving a nick with an unsaturated sugar-phosphate on the 3'-end and a
5'-phosphate (14-16). Traditionally, this AP lyase activity has been
associated with DNA glycosylases such as thymine glycol-DNA glycosylase, formamidopyrimidine-DNA glycosylase, endonuclease V, and
endonuclease VIII (4, 17-20). Subsequent
-elimination of the
3'-terminal sugar-phosphate produces 4-hydroxy-2-pentenal and
3'-phosphate (9) (Fig. 1B), which must be further processed to generate a 3'-hydroxyl required for DNA polymerase gap-filling synthesis. The single nucleotide gap is filled by DNA polymerase I in
E. coli and by DNA polymerase
(
-pol) in mammalian
cells (21-23). Since mammalian cells are known to contain multiple
species of DNA ligase with broad substrate specificity, one of these
enzymes completes the BER pathway by sealing the nicked DNA (24,
25).
DNA polymerase is a 39-kDa monomeric enzyme. Limited proteolysis
and physical studies have shown that
-pol consists of an
independently folded amino-terminal domain of 8 kDa and a
carboxyl-terminal domain of 31 kDa (26-28). The amino-terminal domain
was originally characterized as a single-stranded DNA binding domain.
Subsequently, it was found to possess binding specificity for the
5'-phosphate in gapped DNA (27-29) and a helix-hairpin-helix motif
found in several other DNA repair enzymes (30, 31). Recently, Matsumoto and Kim (32) demonstrated that
-pol catalyzes removal of dRP from AP
endonuclease-incised AP sites via
-elimination, as opposed to
hydrolysis, and that this dRP lyase activity resides in the amino-terminal 8-kDa domain of
-pol. Further evidence that this reaction proceeds via
-elimination was obtained by Piersen et al. (33), who showed that a Schiff base intermediate is formed between the dRP-containing DNA substrate and the enzyme. The Schiff base nucleophile in the 8-kDa domain has been identified as
Lys72 by site-directed mutagenesis (34). Surprisingly, the
dRP lyase activity of
-pol was reported to be
Mg2+-dependent (10, 32), whereas the
-elimination reactions described previously for other AP lyases do
not require Mg2+ (11). In the present study, we
investigated the catalytic efficiency and requirements of the
-pol
dRP lyase activity using intact AP site-containing DNA substrates or
substrates that have been preincised with AP endonuclease. The findings
are discussed in the context of the NMR and crystal structures of the
-pol 8-kDa domain and the requirements of
-elimination chemistry
in the dRP lyase reaction.
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EXPERIMENTAL PROCEDURES |
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Materials--
High pressure liquid chromatography-purified
oligodeoxynucleotides were obtained from Operon Technologies, Inc.
[-32P]ddATP (3000 Ci/mmol) was purchased from Amersham
Pharmacia Biotech. Terminal deoxynucleotidyltransferase was from
Promega. Recombinant human
-pol, the amino-terminal 8-kDa domain,
and the carboxyl-terminal 31-kDa domain were overexpressed and purified
as described previously (28, 35). Human AP endonuclease and uracil-DNA
glycosylase with 84 amino acids deleted from the amino terminus were
purified as described (36, 37). Antisera specific to intact
-pol and 8-kDa domain were raised in rabbits (22). Pyridoxal 5'-phosphate (PLP)
was from Sigma. Alan E. Tomkinson (University of Texas Health Science
Center, San Antonio, TX) provided human DNA ligase I that had been
purified from baculovirus-infected insect cells (38).
3'-End Labeling--
A 49-base pair (bp)
oligodeoxyribonucleotide containing uracil at position 21 was labeled
on its 3'-end by terminal deoxynucleotidyltransferase using
[-32P]ddATP and annealed to its complementary strand
by heating the solution at 90 °C for 3 min, followed by slow cooling
to 25 °C. 32P-Labeled duplex oligodeoxynucleotide was
separated from unincorporated [
-32P]ddATP using a
Nensorb-20 column according to the manufacturer's suggested protocol.
The radiolabeled oligodeoxynucleotide was lyophilized, resuspended in
H2O, and stored at
30 °C.
Preparation of DNA Substrates for AP Lyase and dRP Lyase Assays-- 32P-Labeled uracil-containing duplex oligodeoxynucleotide (62.5 nM) was pretreated for 20 min at 37 °C with uracil-DNA glycosylase (10 nM) in 100 µl of buffer containing 70 mM Hepes, pH 7.4, 0.5 mM EDTA, and 0.2 mM dithiothreitol. Due to the labile nature of the AP endonuclease-treated DNA, the DNA substrate was prepared just prior to the dRP lyase assay.
Enzyme Assays--
AP lyase activity was determined in a
reaction mixture (10 µl) that contained 50 mM Hepes, pH
7.4, 2 mM dithiothreitol, with or without 5 mM
MgCl2, and 20 nM 32P-labeled
double-stranded oligodeoxynucleotide containing an AP site at position
21. The reaction was initiated by adding an appropriate dilution of
-pol or 8-kDa domain, as indicated in the figure legends, and
incubated for 15 min at 37 °C. After the reaction was terminated,
the product was stabilized by the addition of 2 M
NaBH4 to a final concentration of 340 mM and
incubated for 30 min at 0 °C. The stabilized DNA product was
recovered by ethanol precipitation in the presence of 0.1 µg/ml tRNA
and resuspended in 10 µl of gel loading buffer (95% formamide, 20 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol).
After incubation at 75 °C for 2 min, the reaction products were
separated by electrophoresis in a 20% polyacrylamide gel containing 8 M urea in 89 mM Tris-HCl, 89 mM
boric acid, and 2 mM EDTA, pH 8.8, and visualized by
autoradiography. To quantify the product, gels were scanned on a
Molecular Dynamics PhosphorImager model 450, and the data were analyzed
using ImageQuant software. dRP lyase activity was assayed essentially
as described for the AP lyase reactions described above except that the
uracil-DNA glycosylase-reacted DNA substrate was further treated with
AP endonuclease in the presence of 5 mM MgCl2
to create a substrate containing a 5'-incised AP site (Fig.
1).
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Effect of pH on dRP Lyase Activity of -Pol and the 8-kDa
Domain--
The dRP lyase activity measurements were done in a
multiple buffer system containing 25 mM acetic acid, 25 mM MES, and 50 mM Tris. The pH was adjusted
with either HCl or NaOH as appropriate. The ionic strength of this
buffer system is constant in the pH range used in this study. Each
reaction pH at 37 °C was determined using a 400-µl mock-up
reaction mixture.
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RESULTS |
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Assay for Cleavage of AP Site-containing Double-stranded DNA by
Human DNA Polymerase and AP Endonuclease--
Both
-pol and AP
endonuclease cleave double-stranded DNA containing an AP site (4,
13-16, 32). Whereas the
-pol-catalyzed reaction is via
-elimination incising the AP site on the 3'-side of the sugar, AP
endonuclease hydrolyzes the phosphodiester bond 5' to the AP site sugar
(Fig. 2A). To study these
reactions, we utilized a 49-bp oligonucleotide duplex DNA that
contained a uracil residue at position 21. The uracil-containing DNA
strand was 3'-end-labeled with [32P]ddAMP and annealed to
its complementary DNA strand. To generate an AP site, this
32P-labeled duplex DNA was treated with uracil-DNA
glycosylase to quantitatively remove the uracil residue. This results
in an AP site-containing strand where the 32P label is
situated 3' to the lesion (Fig. 2A). The AP site-containing DNA was incubated with either AP endonuclease or
-pol. Since AP
endonuclease incises the AP site DNA strand 5' to the AP site and
-pol incises 3', the AP endonuclease DNA product bears a nick with a
5'-dRP moiety and a 3'-hydroxyl. In contrast, the
-pol-incised DNA
product bears a 3'-dRP moiety and a 5'-phosphate (Fig. 2A).
To resolve the cleaved labeled DNA products bearing either a 5'-dRP
moiety or a 5'-phosphate, the products were stabilized by
NaBH4 reduction at the end of each reaction period, and the incised DNA products were separated by electrophoresis in a 20% denaturing polyacrylamide gel.
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dRP Lyase Releases 5'-Terminal Deoxyribose Phosphate from
Preincised AP Site DNA--
Similar -elimination lyase chemistry
can be used for removal of the 5'-dRP from an AP endonuclease
preincised AP site as with lyase cleavage of an intact AP site, as
illustrated in Fig. 1. To study the dRP release reaction, the DNA
substrate was prepared by pretreating 32P-labeled duplex
DNA containing an AP site with AP endonuclease. The resulting DNA
substrate with a dRP moiety at the 5'-end and a 32P-label
at the 3' terminus (Fig. 3A)
was incubated with
-pol or its amino-terminal 8-kDa domain. As shown
in Fig. 1, the dRP group can be cleaved from the preincised AP site
either by hydrolysis or via a
-elimination reaction.
-Pol is
proposed to catalyze the release of the 5'-dRP moiety from the cleaved
AP site via a
-elimination mechanism, producing
4-hydroxy-2-pentenal-5-phosphate (Fig. 1D). Fig.
3B demonstrates that
-pol and the 8-kDa domain released
dRP from the substrate DNA in a time-dependent manner. The
release of the sugar-phosphate from the 5'-end of the
32P-labeled substrate is visualized by the appearance of a
band migrating approximately one-half nucleotide faster than the
substrate (Fig. 3). The time course of dRP removal was linear for at
least 10 min (Fig. 3C).
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Steady-state Kinetic Parameters of the dRP Lyase Reaction--
To
quantify the kinetic parameters for release of dRP from a preincised AP
site-containing DNA, dRP release was examined as a function of AP site
concentration (Fig. 4). The apparent
Km and kcat for the dRP lyase
activity of -pol were 0.5 µM and 0.075 s
1, respectively (Table I
(top) and Fig. 4A). This results in a catalytic efficiency
(kcat/Km) of 0.15 µM
1 s
1. The dRP lyase
activity of
-pol resides in its amino-terminal 8-kDa domain (32). In
the current study, we did not detect any dRP lyase activity associated
with the carboxyl-terminal 31-kDa domain of
-pol, even with much
higher 31-kDa domain concentrations than that used with intact
-pol
(see below). For the 8-kDa domain, dRP lyase activity was not saturated
at the highest concentration of substrate examined
(i.e. 2 µM). However, the catalytic
efficiency, as measured by
kcat/Km, was 7-fold lower
than that of intact
-pol (Fig. 4B and Table I (top)).
These results suggest that although the 8-kDa domain of
-pol can
perform efficient cleavage of dRP from preincised AP site DNA, the
8-kDa domain requires the 31-kDa domain for full efficiency. Finally,
attempts to restore 8-kDa dRP lyase activity to an efficiency similar
to that of intact
-pol, by supplementing the 8-kDa domain with
purified 31-kDa domain, were unsuccessful (data not shown).
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Steady-state Kinetic Parameters of AP Lyase Using Intact AP Site
DNA as Substrate--
We also determined the kinetic parameters of
-pol and the 8-kDa domain utilizing an intact AP site-containing
duplex DNA as substrate. DNA polymerase
can incise intact AP sites
but does so inefficiently as compared with a preincised AP site (Table I). The results show that while the Km for dRP and
AP lyase activities of
-pol are similar, kcat
is approximately 200-fold lower for the lyase activity on an intact AP
site (Table I, Fig. 4C). As with
-pol, the apparent
activity exhibited by the 8-kDa domain was over 2 orders of magnitude
lower on an intact AP site, as compared with a preincised AP site, when
assayed at similar substrate concentrations (Table I). As with the
preincised AP site with the 8-kDA domain, the elevated
Km for substrate DNA precluded measurement of the
Michaelis constant and kcat.
Cation Dependence of the dRP Lyase Reaction--
To determine
whether the -pol dRP lyase reaction required Mg2+,
uracil-containing 3'-32P-labeled DNA was treated
sequentially with uracil-DNA glycosylase (without Mg2+) and
then with AP endonuclease (with 5 mM MgCl2).
The reaction mixture was then supplemented with the indicated
concentrations of EDTA and/or NaCl. The dRP lyase activity of either
intact
-pol or the 8-kDa domain was assayed by release of dRP from
preincised AP site DNA as described above. The results show that 5 or
10 mM EDTA inhibited
-pol dRP lyase activity (Fig.
5A, lanes
3 and 4). In contrast, activity of the 8-kDa
domain at these EDTA concentrations (5 or 10 mM) was
slightly stimulated (Fig. 5A, lanes 8 and
9). Surprisingly, higher EDTA concentrations (20 and 40 mM) restored the
-pol activity to a level similar to
that in the absence of EDTA (Fig. 5A, compare lane
2 with lanes 5 and 6). Further increases in
EDTA concentration either had no effect or were slightly inhibitory for
the activity of the 8-kDa domain. The inhibitory effect of 5-10
mM EDTA on dRP lyase activity of
-pol was reversed by
25-100 mM NaCl (Fig. 5A, lanes
12-14). The addition of 25-100 mM NaCl to the
reaction mixture did not change the activity of the 8-kDa domain. We
also studied the effect of NaCl, without EDTA, on the dRP lyase
activities of
-pol and the 8-kDa domain (Fig. 5B). The
results show that NaCl, up to 50 mM, had no influence on
the two activities, but concentrations of 200 mM and above
completely abolished activity. Taken together, these results indicate
that Mg2+ is not required for
-pol dRP lyase activity.
EDTA appears to modulate an ionic strength requirement for activity of
the intact protein. EDTA shows inhibitory effects on the dRP lyase
activity of intact
-pol when added in an equimolar ratio to
Mg2+, but this inhibitory effect can be overcome by NaCl.
This effect requires the 31-kDa domain, since EDTA had no influence on
the dRP lyase activity of the 8-kDa domain alone.
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Inhibition of dRP Lyase Activity by PLP and Antibodies to
-Pol--
Protein modification with PLP involves interaction of the
formyl moiety of PLP with free amines on a protein forming a reversible imine or Schiff base. The Schiff base can be reduced to a stable, secondary amine with sodium borohydride. Thus, since Schiff base formation is involved in the dRP lyase reaction catalyzed by
-pol (33), treatment of the enzyme with PLP may be expected to inhibit the
lyase reaction. To test this idea, the dRP lyase reaction was performed
with samples of
-pol and 8-kDa domain that had been treated with
PLP. PLP modification resulted in approximately 95 and 75% inhibition
of the dRP lyase activity of
-pol and the 8-kDa domain, respectively
(Fig. 6C). The results suggest
that a PLP-reactive primary amine group is involved in dRP lyase
catalytic activity. It had been shown earlier that Lys72 in
intact
-pol is modified by PLP (41). Further, NMR studies of the
8-kDa domain have revealed that two lysines in the proposed lyase
active site, Lys72 and Lys60, are
preferentially modified by PLP (42). Thus, our present results on PLP
inhibition of the dRP lyase activity are consistent with earlier
results that
-pol can be PLP-modified. More recently, we have
demonstrated that alanine substitution for Lys72 in the
8-kDa domain resulted in greater than 90% loss of the dRP lyase
activity associated with this domain (34). In addition, the same
mutation in full-length protein resulted in more than 80% loss of dRP
lyase activity without altering DNA synthesis activity (data not
shown).
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Effect of pH on the dRP Lyase Catalytic Efficiency of -Pol and
the 8-kDa Domain--
The relative pH profiles of the
-pol and
8-kDa domain dRP lyase catalytic efficiency were determined using a
multiple buffer system, in order to maintain constant ionic strength
across the pH range (Fig. 7). Apparent
activity was also determined in the absence of enzyme to correct for pH
effects on substrate stability. For the pH range examined, the pH
optimum for
-pol was broad, pH 7-9. The relative pH profile of the
8-kDa domain was also broad, with a pH optimum of 8-9. In relation to
the optimal pH (i.e. pH 8), the 8-kDa domain is less
pH-sensitive at lower pH (below 7). This difference in the pH profiles
suggests that there are changes in the pKa values of
catalytically important residues of the 8-kDa domain when it is removed
from intact enzyme. The low sensitivity of the catalytic efficiency
toward pH and the overall lower catalytic efficiency of the 8-kDa
domain (Table I) are consistent with a critical amine residue being
protonated over the entire pH range. The pH profile of the intact
enzyme suggests that multiple ionizable groups influence lyase
catalytic efficiency. The decline in efficiency with the intact enzyme
at lower pH may reflect protonation of a catalytically important residue, presumably a lysine with a reduced pKa.
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DISCUSSION |
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In addition to BER, a number of alternative routes of enzymatic
excision of the 5'-dRP moiety have been proposed (10). For example, the
mammalian 5' 3' exonuclease DNase IV was shown to release the
5'-terminal dRP residue as part of a small oligonucleotide product (4).
Similarly, the 5'
3' exonuclease activity of E. coli DNA
polymerase I can remove 5'-terminal dRP in the oligonucleotide form
(44). Since these hydrolytic exonucleases remove 5'-dRP as part of a
small oligonucleotide, it is possible that a repair patch size longer
than one nucleotide results from repair in the presence of an
unprocessed 5'-dRP. An alternate BER pathway exhibiting a longer DNA
synthesis patch size (2-6 nucleotides) has been described (43).
However, the predominant BER pathway in mammalian cells involves
incorporation of a single nucleotide where the dRP moiety in the gap
must be removed. Therefore, regulation and coordination of the
enzymatic activities required to complete BER may depend on the removal
or identity of the 5'-sugar-phosphate moiety. This could depend on the
nature of the AP site (i.e. type of base damage or
glycosylase that initiates BER). Our results show that
-pol efficiently removes dRP from the preincised AP site, consistent with
the idea that the dRP lyase activity of
-pol removes the 5'-dRP
during simple BER. These results are also consistent with the recent
observations of Biade et al. (45) and Fortini et al. (43) describing an alternate BER pathway that can utilize a
DNA polymerase other than
-pol. Using nuclear extracts from wild-type and
-pol null mouse fibroblasts, they demonstrated that
the alternate BER pathway repair patch size is 2-6 nucleotides.
Nonenzymatic attack at C1' through a lyase mechanism may be promoted by
basic cellular macromolecules such as polyamines or histones and by
other basic molecules including tripeptides such as Lys-Trp-Lys and
Lys-Tyr-Lys (46). Such nonenzymatic cleavage generally occurs at a much
lower efficiency than that observed with the dRP lyase of -pol (47).
The catalytic efficiency
(kcat/Km) of the
-pol dRP
lyase facilitates our understanding of the rate-determining steps in
BER. For example, we found that kcat of the dRP
lyase of intact
-pol is approximately 10-fold lower than
kcat for DNA synthesis activity (34) and
100-fold lower than kcat of AP endonuclease (36). Assuming that these kcat values are
similar to those operating in BER, these results indicate that the dRP
lyase reaction would be the rate determining step in BER. Thus, there
could be an accumulation of filled gaps (i.e. a
single-nucleotide had been incorporated by
-pol) where the 5'-dRP is
awaiting removal. Alternatively, if dRP removal is a strict requirement
for DNA synthesis and/or ligation in BER, the
-pol dRP lyase
activity could be critical in regulating not only the rate of simple
BER but also alternate BER pathways.
A dRP lyase activity catalyzing the release of 5'-dRP from preincised
AP site-containing DNA was reported by Lindahl and co-workers (10, 13)
in E. coli and later in human cells. They showed that the
reaction was catalyzed by a hydrolytic mechanism that required
Mg2+. In contrast, Matsumoto and Kim (32) showed that a
-pol-mediated dRP lyase reaction was catalyzed via
-elimination.
Surprisingly, this reaction also was
Mg2+-dependent. Historically, the hydrolytic
dRP lyases require Mg2+, whereas AP lyases, which catalyze
via
-elimination, do not require Mg2+ (11). The results
presented here demonstrate that the dRP lyase activity of
-pol
removes 5'-dRP from the preincised AP site without a Mg2+
requirement. Interestingly, the dRP lyase activity of intact
-pol
was inhibited by low concentrations of EDTA but not by high EDTA
concentrations. The EDTA inhibition was reversed by NaCl. The dRP lyase
activity of the
-pol 8-kDa domain was not affected significantly by
the presence of EDTA. These results suggest that there is a
Mg2+-dependent event, not related to dRP
removal, that higher concentrations of Na+ can
"complement" with full-length
-pol. Since the 31-kDa domain has
been shown to change its conformation upon binding of monovalent or
divalent ions (48-50), EDTA may indirectly inhibit the dRP lyase activity of intact
-pol by altering the 31-kDa domain. Our results indicate that the 31-kDa domain of
-pol promotes the dRP lyase activity, but the 31-kDa domain alone has no dRP lyase activity.
Since -pol can incise intact AP sites, this activity may be
important for the cell in removing AP sites in the absence of the major
AP endonuclease. Recently, the Drosophila ribosomal protein
S3 was reported to contain a dRP lyase activity (12) that can process
3' or 5' termini after the initial incision event. Interestingly, the
release of 5'-dRP from the AP endonuclease-cleaved AP site is carried
out by
-elimination, whereas the blocked 3'-hydroxyl generated from
lyase cleavage of an intact AP site is processed through a
Mg2+-dependent hydrolytic mechanism. Therefore,
the Mg2+ dependence for AP site enzymology emerges as a
distinguishing feature, and multiple alternate enzyme activities may be
involved in BER in the absence of the three predominant enzymes (AP
endonuclease,
-pol, and DNA ligase).
Structural characterization of the 8-kDa domain has identified a
structural motif that binds a monovalent metal and interacts with the
DNA backbone (51). This helix-hairpin-helix motif (residues 55-79) is
also observed in endonuclease III, which has both glycosylase and AP
lyase activities. The lyase activity of endonuclease III is through a
-elimination mechanism and results in a 3'-
,
-unsaturated aldehyde and a 5'-phosphate at the termini (Fig. 1A).
Lys120 of endonuclease III has been identified as the
primary amine that forms a Schiff base intermediate (52). Alignment of
the endonuclease III and 8-kDa domain helix-hairpin-helix motifs
suggest that Lys68 of
-pol may be the nucleophilic
residue (51). However, alanine substitution for this lysine did not
influence lyase activity (34). Finally, the use of PLP inhibition and
pH profile analysis of the
-pol and 8-kDa domain dRP lyase
activities provides insight into residues involved in lyase reaction
chemistry. Lys72 is preferentially modified by PLP in both
-pol and the 8-kDA domain (42, 53). Thus, this particular lysine
residue can act as the Schiff base nucleophile attacking PLP at neutral
pH. Site-directed mutagenesis of Lys72 to alanine in the
recombinant 8-kDa domain was found to diminish dRP lyase activity
nearly 2 orders of magnitude, indicating that this residue is the
likely nucleophile that forms the Schiff base intermediate in the
-elimination reaction (34). This residue is observed to coordinate
the 5'-phosphate in a DNA gap in crystal structures of
-pol (51,
54). The decrease in dRP lyase catalytic efficiency at low pH is
consistent with protonation of the lysine nucleophile at low pH. The
broad pH optimum extending into the physiological pH range suggests
that the pKa of the Lys72
-NH2 is diminished significantly. Lys72 is
observed to be part of a lysine-rich pocket on the surface of the 8-kDa
domain (51) that may reduce its pKa. The low
catalytic efficiency and sensitivity of the efficiency of the 8-kDa
domain to pH indicates that the environment of the nucleophile is
altered in the isolated 8-kDa domain. The crystal structure of
-pol
bound to a one-nucleotide gap DNA substrate indicates that the 8-kDa
and the carboxyl-subdomain of the 31-kDa domain interact (54).
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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.
¶ On sabbatical from the Department of Biology, Northeastern University, Boston, MA 02115.
To whom correspondence should be addressed: Laboratory of
Structural Biology National Institute of Environmental Health Sciences, 111 T. W. 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.
1
The abbreviations used are: BER, base excision
repair; -pol, DNA polymerase
; AP, apurinic/apyrimidinic; dRP,
2-deoxyribose-5-phosphate; PLP, pyridoxal 5'-phosphate; bp, base pair;
MES, 4-morpholineethanesulfonic acid; ddATP, dideoxy-ATP; ddAMP,
dideoxy-AMP.
2 E. K. Dimitriadis, M. K. Vaske, R. Prasad, A. E. Tomkinson, M. S. Lewis, and S. H. Wilson, manuscript in preparation.
3 D. K. Srivastava, R. Prasad, B. J. Vande Berg, J. Y. Molina, W. A. Beard, A. E. Tomkinson, and S. H. Wilson, manuscript in preparation.
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REFERENCES |
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