Characterization of an Endonuclease IV 3'-5' Exonuclease
Activity*
Sinéad M.
Kerins,
Ruairi
Collins, and
Tommie V.
McCarthy
From the Department of Biochemistry, University College Cork,
Cork, Ireland
Received for publication, October 21, 2002
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ABSTRACT |
Previous characterization of Escherichia
coli endonuclease IV has shown that the enzyme specifically
cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3'
DNA blocking groups. By contrast, and unlike the major
apurinic/apyrimidinic endonuclease exonuclease III, negligible
exonuclease activity has been associated with endonuclease IV. Here we
report that endonuclease IV does possess an intrinsic 3'-5' exonuclease
activity. The activity was detected in purified preparations of the
endonuclease IV protein from E. coli and from the distantly
related thermophile Thermotoga maritima; it co-eluted with
both enzymes under different chromatographic conditions. Induction of
either endonuclease IV in an E. coli overexpression system
resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the
endonuclease IV AP endonuclease activity. Characterization of the
exonuclease activity showed that its progression on substrate is
sensitive to ionic strength, metal ions, EDTA, and reducing conditions.
Substrates with 3' recessed ends were preferred substrates for the
3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high
and is likely to be significant in vivo.
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INTRODUCTION |
Apurinic and apyrimidinic
(AP)1 sites threaten genetic
stability because they block replication and are mutagenic (1, 2). They
arise in DNA through the spontaneous loss of normal or damaged bases or
through the release of modified or mismatched bases from DNA by DNA
glycosylases (3, 4). The first general step of base excision repair
following base loss is the recognition and cleavage of DNA AP sites by
an AP endonuclease. This is conserved from bacteria to humans.
There are two characterized conserved AP endonuclease families. These
enzymes cleave the DNA backbone immediately 5' of an AP site,
generating a 5'-deoxyribose phosphate group and a 3' deoxyribose-hydroxyl group that primes DNA repair synthesis (4). Removal of the 5'-deoxyribose phosphate group by a 5'-deoxyribose phosphodiesterase is considered to create a single-nucleotide gap, and
repair is completed by DNA polymerase-mediated DNA resynthesis and
rejoining via DNA ligase (5, 6). The first enzyme family is typified by
exonuclease (Exo) III from Escherichia coli (7, 8) and the
homologous APE-1 enzyme in humans (9), which are major AP endonucleases
in these organisms. The second conserved AP endonuclease family is
typified by E. coli endonuclease (Endo) IV (10, 11) and
includes the APN-1 protein from Saccharomyces cerevisiae
(12) and Schizosaccharomyces pombe (13) and the CeAPN1 gene
from the nematode Caenorhabditis elegans (14). In E. coli, Endo IV expression is induced by superoxide anion generators (15), but in S. cerevisiae, APN-1 is the predominant
constitutive AP endonuclease.
Genetic studies indicate that Exo III and Endo IV have
overlapping but distinctive repair specificities in vivo. In
E. coli Exo III is encoded by the xth gene (16)
and is a constitutive enzyme accounting for 80-90% of the total AP
activity in the cell. Endo IV is encoded by the nfo gene
(17) and accounts for 5-10% of the total cellular AP activity (8).
Exo III is a divalent metal ion-dependent enzyme and is
inactivated by metal chelating agents (18). In contrast, the AP
activity of E. coli Endo IV is resistant to inactivation by
EDTA in normal assay conditions (11). Exo III and Endo IV also have a
3'-phosphatase and a 3'-repair phosphodiesterase in common. These
activities are responsible for removing a multitude of blocking groups,
including 3'-phosphoglycolate and 3'-phosphate, that are present at
single-stranded breaks in DNA induced by oxidative agents (8, 18). Endo
IV is the only known enzyme that is active against damaged nucleotides
with bases in the
configuration (19). Exo III has a 3'-5'
exonuclease activity, the functional significance of which is unknown
(20). Even though E. coli Endo IV is a minor AP
endonuclease, its expression can be induced more than 20-fold by
superoxide-generating agents, such as paraquat (15), which thus
enhances the capability of the cells for repairing oxidative DNA damage
or damage that is refractory to enzymatic processing by Exo III.
nfo-like endonucleases have also been reported to nick DNA
on the 5' side of various oxidatively damaged bases (21).
The Endo IV active site contains a trinuclear zinc center that is
ligated by conserved protein side chains that cluster at the center of
a deep, crescent shaped groove (22). Biochemical experiments have
suggested a role for manganese (9), although the crystal structure of
the Endo IV complexed with DNA indicates that manganese is not needed
for activity (22). Two of the zinc atoms are partially buried in the
enzyme, whereas the third atom is relatively accessible. The high
resolution structures suggest that the geometry of the Endo IV
trinuclear zinc cluster is exquisitely tuned for cleaving
phosphodiester bonds, with all three zinc ions participating in
catalysis (8, 22). To date, no significant exonuclease activity has
been associated with Endo IV (10, 11). Here we report detection and
characterization of a significant Endo IV 3'-5' exonuclease activity.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]ATP (3000 Ci/mmol) was
obtained from Amersham Biosciences. E. coli Endo IV was
obtained from Epicentre Technologies and Fermentas and generously
supplied by Bruce Demple (Harvard University, Boston, MA).
Overexpression of the E. coli and Thermotoga maritima Endo IV
Genes--
The E. coli Endo IV gene (Ec-Endo IV) was
amplified by PCR from E. coli K12 genomic DNA using
the following forward and reverse primers:
5'-ATGAAATACATTGGAGCGCA-3' and 5'-GGCTACCCGCTTTTTCAGT-3', designed from the published sequence accession number M22591 (16). The
T. maritima Endo IV gene (Tm-Endo IV) was amplified from
T. maritima genomic DNA using the primers
5'-ATGATAAAAATAGGAGCTCACA-3' and
5'-ATCGACCTCTATACCGAATT-3' designed from sequence data obtained from the Institute for Genomic Research (www.tigr.org). The
reverse primers used for the amplification of both genes were designed to exclude the native stop codon. Amplification of the genes was performed by PCR (95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min for 35 cycles followed by 72 °C for 10 min) in a reaction volume of 100 µl containing 300 nM of each primer, 200 µM of each deoxynucleoside triphosphate, 500 ng of
T. maritima or E. coli genomic DNA, and 2.6 units
of expand high fidelity DNA polymerase (Roche Molecular Biochemicals)
in the supplied buffer. The amplified products of the anticipated
sizes, 855 bp for the Ec-Endo IV gene and 861 bp for the Tm-Endo IV
gene, were generated and cloned in frame with a C-terminal His tag in
the expression vector pBAD-TOPO and transformed into E. coli
TOP10 cells according to the manufacturer's instructions (Invitrogen).
Purification of E. coli and T. maritima Endonuclease
IV--
E. coli TOP10 cells harboring the pBAD-TOPO/Ec-Endo
IV or Tm-Endo IV constructs were grown overnight at 37 °C in 10 ml
of LB containing 50 µg/ml ampicillin (LB-amp). This overnight culture was used to inoculate a 1-liter LB-amp culture, which was grown to an
A600 of 0.5, at which point arabinose was
added to a final concentration of 0.2% w/v. The cells were allowed to
grow for a further 6 h at 37 °C and were then collected by
centrifugation at 3000 × g for 20 min at 4 °C.
Following this they were resuspended in 20 ml of buffer A (50 mM phosphate buffer, pH 7.5, 500 mM NaCl, 20 mM imidazole) and lysed by sonication (four 10-s bursts at a medium setting). The crude extract was clarified by centrifugation a
12,000 × g for 30 min at 4 °C. Batch application of
the supernatant to 1 ml of Pro-Bond resin (Invitrogen),
pre-equilibrated with buffer A was performed at 4 °C for 1 h
with gentle agitation. Unbound proteins were collected in the flow
through, which was followed by two subsequent 10-ml washes with buffer
A and then buffer B (50 mM phosphate buffer, pH 7.5, 50 mM NaCl, 35 mM imidazole). The Ec-Endo IV and
Tm-Endo IV His-tagged proteins were collected as 0.5-ml fractions
following application of buffer C (50 mM phosphate buffer,
pH 7.5, 50 mM NaCl, 100 mM imidazole). Fast
protein liquid chromatography was then used to purify the proteins
further. Fractions containing the 35.5-kDa Ec-Endo IV fusion protein
fractions were applied to a MonoQ (DIONEX), whereas the 36.9-kDa
Tm-Endo IV fusion protein fractions were applied to a MonoS HR 5/5
column (Pharmacia Corp.). The proteins were eluted with a 20-ml linear
gradient from buffer D (10 mM Hepes, pH 7.2) to buffer D
containing 1 M NaCl at a flow rate of 1 ml/min. Fractions
of 0.5 ml were collected, and samples were analyzed on a 15%
SDS-polyacrylamide gel electrophoresis and visualized by staining with
Coomassie Brilliant Blue. The Ec-Endo IV fusion protein was eluted with
a salt concentration of 337-375 mM, whereas the Tm-Endo IV
fusion protein was eluted with a salt concentration of 600-625
mM. For comparison of induced and noninduced extracts,
E. coli TOPO10 cells harboring the empty inducible pBADTOPO
expression vector or the vector bearing the E. coli or
T. maritima Endo IV genes were inoculated into 10 ml of LB
medium containing 100 µg/ml ampicillin and were grown overnight at
37 °C. 1 ml of the overnight cultures were used to inoculate 100 ml
of LB-amp medium and were grown at 37 °C to an optical density of
0.5 at 600 nm. Half of each culture was induced with 0.2% arabinose,
and all cultures were grown for an additional 6 h. Crude extracts
were then prepared and heat-treated at 65 °C for 7 min as described
previously (23). Following 30 min on ice, the heat-treated extracts
were clarified by centrifugation at 12,000 × g for 30 min at 4 °C.
DNA Substrates--
Oligonucleotides were purchased from Sigma
Genosys apart from the oligonucleotide containing a central synthetic
(tetrahydrofuranyl) AP site, which was purchased from Integrated DNA
Technologies (Coralville, IA). 5' end labeling was performed in a
50-µl reaction at 37 °C for 30 min using 20 units of T4
polynucleotide kinase (New England Biolabs) and
[
-32P]ATP (3000Ci/mmol) in the kinase buffer supplied
by the manufacturer. Nucleotides were then removed from the reaction
mix using a mini-quick spin oligo column (Roche Molecular
Biochemicals). The end-labeled oligonucleotide and its complement were
mixed in equimolar quantities and annealed by heating to 95 °C for 5 min followed by cooling to room temperature. The following substrates
were used in enzymatic assays. SubSS-19AP was the single-stranded
oligonucleotide
5'-GGCGAACGAGACGAGGG(C/A)(P/G)CTGGAAAGG-3' bearing
the synthetic AP site, tetrahydrofuranyl. The double-stranded form of the substrate, subDS-19AP, was generated by annealing with its
complement 3'-CCGCTTGCTCTGCTCCCGTCGACCTTTCC-5'. SubSS-18R was
the single-stranded oligonucleotide 5'-GGCGAACGAGACGAGGGC-3'. The
double-stranded form of the substrate, subDS-18R, was generated by
annealing subSS-18R with the oligonucleotide
3'-CCGCTTGCTCTGCTCCCGTCGACCTTTCC-5'. Substrate subDS-20R
was generated by annealing the oligonucleotides 5'-ATGTAACATCTTGCAGTCGG-3' and
3'-TACATTGTAGAACGTCAGCCTATTCACGAA-5'. Substrate subDS-20O was
generated by annealing the oligonucleotides 5'-ATGTAACATCTTGCAGTCGG-3' and 3'TACATTGTAGAACGTCAG-5' and
substrate subDS-30B was generated by annealing
5'-ATGTAACATCTTGCAGTCGGATAAGTGCTT-3' and
3'-TACATTGTAGAACGTCAGCCTATTCACGAA-5'.
Enzymatic Assays--
The assays were performed using 500 fmol
of the 5' end-labeled substrate in a total volume of 25 µl. All of
the reactions were incubated at either 37 °C (Ec-Endo IV) or
60 °C (Tm-Endo IV) for 15 min. Prior to optimization, all of the
assays were carried out in 10 mM Tris/HCl, pH 8.3, 10 mM KCl. For determination of optimal pH, the assays were
performed in buffers (25 mM) of varying pH: sodium
acetate/acetic acid, pH 5, MES/NaOH with pH 5.5, 6.0, or 6.5, Tris/HCl
with pH 7.0, 7.5, 8.0, or 8.5, and glycine/NaOH with pH 9.0, 9.5, 10.0, or 10.5. Following pH optimization, all of the subsequent reactions
were in 25 mM glycine/NaOH, pH 9.0. The reactions were
stopped by transferring tubes to ice and adding an equal volume of
loading solution (98% formamide, 10 mM EDTA, 0.5% xylene
cyanole, and 0.5% bromphenol blue). The reaction products were
analyzed by denaturing gel electrophoresis (20% polyacrylamide, 7 M urea) gels and quantified by phosphorimaging analysis
using ImageQuant Software (Molecular Dynamics, Inc.).
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RESULTS |
Overexpression and Purification of E. coli and T. maritima
Endonuclease IV--
The E. coli and T. maritima
Endo IV genes were amplified from genomic DNA using primers designed
from the published sequence and cloned into the pBAD-TOPO expression
vector under the control of the E. coli arabinose-inducible
pBAD promoter. The primers were designed so that the Endo IV open
reading frames were cloned into the vectors in frame with a N-terminal
leader sequence, encoding a 1.5-kDa polypeptide, and a C-terminal
sequence, encoding a 3-kDa polypeptide, which includes the V5 epitope
tag and a polyhistidine region. Following growth of E. coli
strains harboring the pBAD-TOPO-Ec-Endo IV and pBAD-TOPO-Tm-Endo IV and
induction of the fusion genes by arabinose, crude extracts were
prepared, and the 35.5-kDa Ec-Endo IV and 36.9-kDa Tm-Endo IV fusion
proteins were purified by immobilized metal affinity chromatography.
Pooled samples bearing the eluted proteins of the expected sizes were
further purified using ion exchange chromatography, anion exchange
(MonoQ) for the E. coli protein, and cation exchange MonoS
for the (T. maritima) protein. Analyses of the purified
fractions by SDS-PAGE and Coomassie Blue staining showed that the
Ec-Endo IV and the Tm-Endo IV fusion proteins were >95% pure (Fig.
1A).

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Fig. 1.
Exonuclease activity associated with E. coli and T. maritima Endo IV
proteins. A, SDS-PAGE (15%) analysis of MonoQ and
MonoS purified fractions of E. coli and T. maritima Endo IV samples used for the exonuclease assay. Following
electrophoresis the proteins were visualized by staining with Coomassie
Brilliant Blue. Lane 1, protein standard marker; lane
2, 3.5 µg of Ec-Endo IV; lane 3, 1.7 µg of Tm-Endo
IV. B, the 5' end-labeled substrate subDS-18R (500 fmol) was
incubated with the purified E. coli or T. maritima Endo IV proteins. Lane 1, subDS-18R, no enzyme
added; lanes 2 and 3, subDS-18R incubated with
100 ng of Ec-Endo IV or 100 ng of Tm-Endo IV, respectively.
n, nucleotides.
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Evaluation of the Exonuclease Activity--
Exonuclease activity
in the MonoQ and MonoS Ec-Endo IV and Tm-Endo IV fractions was
initially evaluated by incubation of the enzymes with a double-stranded
5' end-labeled 3' recessed 18-nt substrate (subDS-18R) at 37 and
60 °C, respectively. The buffer used in this initial assay was
different from that used previously in reports investigating Endo IV
activity (11). In particular, EDTA and DTT were absent, and the salt
concentration was low. Analysis of samples incubated with either Endo
IV revealed multiple bands smaller in size than the labeled 18-nt
fragment and indicating the presence of exonuclease activity. The band
sizes decreased approximately one base at a time, consistent with the
presence of a 3'-5' exonuclease activity (Fig. 1B). The fact
that significant exonuclease activity was present in the MonoQ-purified
Ec-Endo IV and the MonoS-purified Tm-Endo IV showed that the activity was co-purified with the two Endo IV proteins under very different ion
exchange conditions and indicated that the activity might be intrinsic
to the Endo IV enzymes. In agreement with this, the exonuclease
activity associated with the purified Ec-Endo IV sample has a heat
stability (stable after incubation at 65 °C for 5 min) similar to
that described for the AP endonuclease activity of the Ec-Endo IV (data
not shown) (23). The possibility that the exonuclease activity was an
arabinose-inducible E. coli gene was investigated. E. coli harboring the empty inducible pBAD-TOPO expression vector or
the vector bearing the Ec-Endo IV or Tm-Endo IV genes were grown under
identical conditions and induced with arabinose. Crude cell extracts
were preincubated at 65 °C for 7 min and assayed for exonuclease
activity. A low level of background activity was detected in cells
harboring the empty vector, but no significant difference in activity
was observed between extracts from noninduced and induced cells (Fig.
2A). Extracts from noninduced cells harboring the Endo IV genes had levels of exonuclease activity similar to that observed in the control extracts. By contrast, significant exonuclease activity was present in extracts from the
induced cells harboring either Endo IV gene, confirming that the
activity is associated with the Endo IV proteins (Fig.
2A).

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Fig. 2.
Induction of exonuclease activity in E. coli harboring the Endo IV gene under an inducible
promoter. Crude cell extracts were prepared from E. coli harboring the pBAD-TOPO expression vector (control) or the
vector bearing the Ec-Endo IV or Tm-Endo IV genes after induction for
6 h with 0.2% arabinose. Following heat treatment at 65 °C for
7 min, the extracts (1 µl) were incubated with the 5' end-labeled
subDS-18R (500 fmol) (A) or the 5' end-labeled AP containing
substrate subDS-19AP (500 fmol) (B). Lanes 1, no
extract added; lanes 2 and 3, noninduced and
induced control extract; lanes 4-7, noninduced and induced
extracts from cells harboring the vector bearing the Ec-Endo IV or
Tm-Endo IV genes, respectively. n, nucleotides.
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The natural substrate for the Endo IV enzymes is an AP site within a
double-stranded DNA context. To determine whether the exonuclease
activity was active at cleaved AP sites, a 5' end-labeled double-stranded 29-nt bearing a central synthetic (tetrahydrofuranyl) AP site at position 19 (subDS-19AP) was incubated with heat-treated extracts prepared from noninduced and induced cells harboring the
plasmids, pBAD-TOPO, pBAD-TOPO-Ec-Endo IV, or pBAD-TOPO-Tm-Endo IV.
Cleavage at the AP site in this substrate generates two primary cleavage products: an end-labeled 18-nt fragment and an unlabelled 11-nt fragment. Significant AP endonuclease activity was present in
each extract and the substrate was cleaved to completion in each case.
The 18-nt primary cleavage product (PCP) was further digested in all
cases. For extracts prepared from cells harboring the empty vector or
from noninduced cells bearing the vectors with the Endo IV genes,
digestion was predominantly by one nucleotide. By contrast, exonuclease
digestion of the 18-nt PCP by the extracts from the induced cells
bearing the vector with either Endo IV gene was much more extensive
(Fig. 2B). The 18-nt PCP was almost entirely degraded, and
the 17-, 16-, and 15-nt products were clearly visible. To confirm the
size of cleavage fragments, cleavage products were analyzed by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry. The sizes generated were consistent with an 18-nt PCP and
17-, 16-, and 15-nt degradation products (data not shown).
A further control was carried out to investigate the possibility that
the exonuclease activity was a contaminant protein that binds to
non-Endo IV parts of the purified Endo IV fusion proteins. Essentially
a uracil DNA glycosylase gene from T. maritima was cloned
into the pBADTOPO expression vector and induced and purified in an
identical fashion to the E. coli and T. maritima
Endo IV genes/proteins. Exonuclease activity above background levels
was not detected when the purified glycosylase from T. maritima was incubated at either 37 or 60 °C with either
substrate (data not shown).
Purified E. coli Endo IV proteins were obtained from
different sources and assayed for exonuclease activity using the
double- and single-stranded substrates, subDS-19AP and subSS-19AP,
respectively. All of the Endo IV proteins cleaved the substrates at the
AP site and exhibited progressive exonuclease activity of varying
levels on the double-stranded substrate
(Fig. 3 and Table
I). Quantitation of the level of cleavage
at the AP site in the substrates showed that the AP activity of the
Endo IV enzymes was almost 2-fold more active on double-stranded DNA in
comparison with single-stranded DNA (Table I). Some exonuclease
activity was detected on the single-stranded substrate, but the extent
of 3'-5' digestion of the 18-nt fragment was significantly less than
that observed for the double-stranded substrate. In addition, the 18-nt
PCP generated from cleavage of the AP site on the single-stranded
substrates was not generally digested by more than one nucleotide in a
3'-5' direction by the exonuclease activity (Fig. 3). Exonuclease
digestion of the 18-nt PCP resulting from cleavage of the AP site in
the double-stranded substrate was substantial. For example, in the case
of the purified Ec-Endo IV MonoQ fraction, when the AP site was cleaved
to ~73%, 73% of product was expected as the 18-nt PCP. However,
only ~33% was present, indicating that ~55% of the 18-nt PCP was
further digested by at least one nucleotide (Table I).

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Fig. 3.
Exonuclease activities of Endo IV proteins
from different sources. The 5' end-labeled substrates, subDS-19AP
(500 fmol; lanes 1-6) and subSS-19AP (500 fmol; lanes
7-12) were incubated with different preparations of Endo IV
proteins. Lanes 1 and 7, no enzyme added;
lanes 2 and 8, 100 ng of the Ec-Endo IV MonoQ
fraction; lanes 3 and 9, 2 units of Ec-Endo IV
(Epicentre); lanes 4 and 10, 2 units of Ec-Endo
IV (Fermentas); lanes 5 and 11, 40 ng of
Ec-EndoIV (Demple); lanes 6 and 12, 100 ng of the
Tm-EndoIV MonoS fraction. n, nucleotides.
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Table I
Exonuclease activity of Endo IV from different sources on substrates
subDS-19AP and subSS-19AP
Assays were performed to determine the exonuclease activity of Endo IV
(EIV) proteins from E. coli and T. maritima.
E. coli Endo IV was purchased from Epicentre (Epi) or
Fermentus (Fer), supplied from Demple (Dem), or purified in this work
(MonoQ sample). Endo IV was also purified from T. maritima
(MonoS sample; this work). For experimental details see legend to Fig.
3. The numbers shown refer to percentages of samples at different
product sizes.
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Characterization of the Exonuclease Activity--
The effect of pH
on the exonuclease activity of E. coli Endo IV was
characterized. A larger amount of enzyme was used for this
characterization to facilitate examination of the effect of pH on both
the activity and extent to which the enzyme progressed on the
substrate. The E. coli Endo IV displayed both AP activity and exonuclease activity over a wide pH range with optimal exonuclease activity at pH 9.0 (Fig. 4A).
At this pH, there was complete cleavage of the AP site in
the substrate. In the absence of exonuclease activity, the 18-nt PCP
should account for 100% of the product. At pH 9.0, the 18-nt PCP only
accounted for 11% of the cleaved product. The smallest digestion
products were detectable at this pH, showing that the enzyme progressed
furthest on the substrate at this pH. Both the exonuclease activity and
the extent of progression on the substrate decreased as pH was
increased above 9.0 (Fig. 4A). Prior to pH optimization, all
of the exonuclease assays were performed in a reaction mixture
containing 10 mM Tris/HCl, pH 8.3, 10 mM KCl.
Following pH optimization, all of the subsequent assays were performed
using 25 mM glycine-NaOH buffer, pH 9.0.

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Fig. 4.
Effect of pH and NaCl on the exonuclease
activity of E. coli Endo IV protein. The 5'
end-labeled substrate subDS-19AP (500 fmol) was incubated with the
MonoQ fraction of Ec-Endo IV under various pH or NaCl conditions.
A, lanes 1-12, substrate was incubated with 600 ng of Ec-Endo IV at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, and 10.5, respectively. B, substrate was incubated
with 100 ng of Ec-Endo IV protein and varying NaCl concentrations.
Lane 1, no Endo IV added; lanes 2-14, NaCl added
to reaction mix to a concentration of 0.0, 0.05, 0.1, 0.15, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, and 1 M, respectively.
n, nucleotides.
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The Endo IV exonuclease activity was very sensitive to the salt
concentration, with little activity observed at NaCl concentrations of
>150 mM (Fig. 4B). Similar results were
obtained for KCl (data not shown). In contrast, the AP endonuclease
activity of Endo IV was active at low and high salt concentrations with
>95% substrate cleavage occurring in the absence of added NaCl and
~75% cleavage occurring in the presence of 1 M NaCl
(Fig. 4B).
The effect of magnesium, the metal ions associated with Endo IV (zinc
and manganese) (9), and EDTA on the exonuclease activity was analyzed
using both substrates (Fig. 5). 100 ng of
the Ec-Endo IV cleaved ~65% of the AP sites in the subDS-19AP
substrate, whereas in the presence of MgCl2,
MnCl2, ZnCl2, MgSO4,
MnSO4, and ZnSO4, cleavage was ~95, 67, 93, 90, 90, and 93%, respectively. It was difficult to estimate
exonuclease activity using this substrate because the level of AP site
cleavage varied. However, it was clear that the exonuclease activity
was most progressive in the presence of zinc (Fig. 5). Progression of
the enzyme on the substrate was also high in the presence of
MnSO4 but not in the presence of MnCl2.
Although the presence of MgCl2 or MgSO4
appeared to stimulate AP activity, the progression of the exonuclease
was lower than that for ZnCl2, ZnSO4, or
MnSO4. Interestingly in the presence of EDTA, the highest
level of digestion of the 18-nt PCP was observed, but progression was
lowest. Overall, the metal ion effects were reproducible; however,
significant variation was observed between experiments. The reason for
this is unclear.

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Fig. 5.
Effect of metal ions on the E. coli Endo IV exonuclease activity. The 5' end-labeled
substrate subDS-19AP (500 fmol) was incubated with 100 ng of the MonoQ
fraction of Ec-Endo IV with various metal ions (2 mM) or
EDTA (2 mM). Lane 1, no Endo IV added;
lane 2, Endo IV added; lane 3, with
MgCl2; lane 4, with MnCl2;
lane 5, with ZnCl2; lane 6, with
MgSO4; lane 7, with MnSO4;
lane 8, with ZnSO4; lane 9, EDTA
added. n, nucleotides.
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The effect of the reducing agent DTT on the exonuclease activity of the
Endo IV was investigated (Fig. 6).
Addition of DTT to the reaction mixture reduced the AP activity of the
enzyme by 2-20% depending on the buffer used and the concentration of agent. Addition of 1 mM DTT to the standard reaction
containing subDS-19AP and Endo IV in the glycine/NaOH reaction buffer,
pH 9.0, did not appear to significantly alter the level of 18-nt PCP
generated. However, in the presence of DTT, the number and intensity of
fragments smaller than 17 nt were much lower than in the absence of
DTT. This indicates that the progression of the exonuclease on the
substrate was impaired by the DTT. The addition of the same amount of
DTT to the standard reaction in Tris/HCl buffer, pH 9.0, had less
effect on the progress of the exonuclease activity. The inhibitory
effect was much more pronounced in 100 mM DTT. Using a
Tris/HCl buffer, pH 7.6, or a Hepes/KOH buffer, pH 7.6, no inhibitory
effect on exonuclease activity was observed in the presence of 1 mM DTT, but impairment progression of the exonuclease was
high at a 100 mM concentration (Fig. 6).

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Fig. 6.
Effect of DTT in different buffers on the
exonuclease activity of E. coli Endo IV. The 5'
end-labeled substrate subDS-19AP (500 fmol) was incubated with 100 ng
of the MonoQ fraction of Ec-Endo IV in different reaction buffers with
various additions to each buffer. Lanes 1-3, 50 mM glycine/NaOH reaction buffer, pH 9.0, with no enzyme
added, enzyme added, and enzyme plus 1 mM DTT added,
respectively; lanes 4-7, 10 mM Tris/HCl
reaction buffer, pH 9.0, with no enzyme added, enzyme added, enzyme
plus 1 mM DTT, and enzyme plus 100 mM DTT
added, respectively; lanes 8-11, 10 mM Tris/HCl
reaction buffer, pH 7.6, with no enzyme added, enzyme added, enzyme
plus 1 mM DTT, and enzyme plus 100 mM DTT
added, respectively, lanes 12-15, 25 mM
Hepes/KOH reaction buffer, pH 7.6, with no enzyme added, enzyme added,
enzyme plus 1 mM DTT, and enzyme plus 100 mM
DTT added, respectively. n, nucleotides.
|
|
Inhibition of the Exonuclease Activity--
Ec-Endo IV has
previously been characterized extensively. However, a significant
exonuclease activity associated with the enzyme has not been reported.
In the majority of characterizations, EDTA, DTT, and KCl have been
included in reaction buffers (10, 11). The effect of these agents,
singularly and combined, on the exonuclease activity of the Endo IV was
investigated (Fig. 7). The addition of
EDTA (1 mM), DTT (1 mM), or KCl (100 mM) to the standard reaction inhibited the exonuclease
activity significantly but not completely. The addition of all three
agents to the reaction mixture inhibited the activity to the extent
that only the 18-nt PCP of the AP endonuclease activity was
detected.

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Fig. 7.
Inhibition of the exonuclease activity of
E. coli Endo IV protein. The 5'
end-labeled substrate subDS-19AP (500 fmol) was incubated with 100 ng
of the MonoQ fraction of Ec-Endo IV with various added reagents. The
standard reaction contains 25 mM glycine/NaOH, pH 9.0, using 100 ng of Endo IV and 500 fmol of subDS-19AP. Lane 1,
no enzyme; lane 2, with enzyme; lane 3, 1 mM EDTA added; lane 4, 1 mM DTT
added; lane 5, 100 mM KCl added; lane
6, 1 mM EDTA and 1 mM DTT added;
lane 7, 1 mM EDTA, 1 mM DTT, and 100 mM KCl added. n, nucleotides.
|
|
Determination of the Relative Activity of the Exonuclease Activity
of Ec-Endo IV on Different Substrates--
To determine the relative
activity of the exonuclease activity of Ec-Endo IV on different
substrates, 100 ng of the enzyme was incubated with 20 and 18-nt duplex
oligonucleotides with 3' recessed ends, a 30-nt duplex substrate with
blunt ends, and a 20-nt duplex substrate with a 3' overhang (Fig.
8). Comparison of the extent of digestion
of the substrates showed that ~20% of the labeled 20-nt
oligonucleotide of subDS-20R was digested by one nucleotide or more,
and more than 45% of the 18-nt oligonucleotide of subDS-18R was
digested. By contrast, the activity on the labeled 30-nt
oligonucleotide of the substrate subDS-30B, was significantly less at
~12%, and the enzyme had little or no activity on 3' overhangs (Fig.
8). Digestion of the 18-nt PCP of subDS-19AP was greatest, with ~72%
of the 18-nt product digested by one or more nucleotides.

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Fig. 8.
Exonuclease activity of E. coli
Endo IV on different DNA substrates. The MonoQ-purified
fraction of Ec-Endo IV was incubated with 500 fmol of each of the 5'
end-labeled DNA substrates. Lanes 1 and 2,
subDS-20R (3' recessed) without and with enzyme respectively;
lanes 3 and 4, subDS-18R (3' recessed) without
and with enzyme, respectively; lanes 5 and 6,
subDS-30B (blunt ended) without and with enzyme, respectively;
lanes 7 and 8, subDS-20O (3' overhang) without
and with enzyme respectively; lanes 9 and 10,
subDS-19AP without and with enzyme, respectively. n,
nucleotides.
|
|
Comparison of the Relative AP Endonuclease and Exonuclease Activity
of Endo IV--
The relative AP endonuclease and exonuclease
activities of Ec-Endo IV were compared (Fig.
9 and Table
II) using both the single- and
double-stranded AP substrates, subSS-19AP and subDS-19AP, respectively.
The level of exonuclease activity on the single-stranded substrate was
low. For this substrate, 78.6% of substrate remained uncleaved after
incubation with 100 ng of Endo IV under the conditions used. Of the
21.4% of cleaved product, the single-stranded 18-nt PCP accounted for
~17.2%, and a 17-nt product accounted for ~4%. This shows that
the 18-nt PCP generated was subsequently digested further by one
nucleotide to a level of ~20%. Additional smaller products were not
observed. Thus, exonuclease activity on the single-stranded substrate
is relatively low. By comparison, the level of exonuclease activity on
the double-stranded substrate was significantly higher. Only 40% of
substrate remained uncleaved after incubation with 100 ng of Endo IV.
Of the 60% of cleaved product, the 18-nt PCP accounted for ~28%, a
17-nt product accounted for ~23%, a 16-nt product accounted for 4%,
and additional smaller products were visible.

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Fig. 9.
Comparison of the AP endonuclease and
exonuclease activity of E. coli Endo IV. 500 fmol
of the 5' end-labeled single and double-stranded substrates, subSS-19AP
(lanes 1-11) and subDS-19AP (lanes 12-22) were
incubated with increasing amounts of the MonoQ fraction of Ec-Endo IV.
Lanes 1 and 12, no Endo IV added; lanes
2 and 13, 0.2 ng; lanes 3 and 14,
0.4 ng; lanes 4 and 15, 0.8 ng; lanes
5 and 16, 1.6 ng; lanes 6 and 17,
3.2 ng; lanes 7 and 18, 6.4 ng; lanes
8 and 19, 12.8 ng; lanes 9 and
20, 25.6 ng; lanes 10 and 21, 51.2 ng;
lanes 11 and 22, 100 ng. n,
nucleotides.
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Table II
Comparison of the AP endonuclease and exonuclease activity of E. coli
Endo IV
For experimental details see legend to Fig. 9. The numbers shown refer
to percent of samples at different product sizes.
|
|
 |
DISCUSSION |
Previous characterization of Endo IV has shown that the enzyme
specifically cleaves the DNA backbone at AP sites and also removes 3'
DNA blocking groups such as 3' phosphates, 3'phosphoglycolates, and 3'
,
-unsaturated aldehydes that arise from oxidative base damage and
the activity of combined glycosylase/lyase enzymes (1, 2, 4). By
contrast, and unlike the major AP endonuclease Exo III, negligible
exonuclease activity has been associated with Endo IV. The results
reported here show that Endo IV does possess an intrinsic 3'-5'
exonuclease activity; the activity was detected in purified
preparations of the Endo IV protein from E. coli and from
the distantly related thermophile T. maritima; it co-eluted with both enzymes under different chromatographic conditions; induction
of either Endo IV in an E. coli overexpression system resulted in induction of the exonuclease activity; and the E. coli exonuclease activity had similar heat stability to the Endo IV AP endonuclease activity.
Considering that Ec-Endo IV is a highly characterized and widely
explored enzyme, we sought to identify the reason that this activity
was not detected previously. Characterization of the exonuclease
activity was carried out using a variety of substrates under a variety
of conditions. The Endo IV exonuclease was active over a broad pH range
with labeled fragments lower than 5 nt detectable at the optimum pH,
indicating that the exonuclease progresses extensively on the substrate
and is active on substrates with small duplexed regions. The Endo IV
exonuclease activity was very sensitive to the ionic strength of the
reaction mixture. As the salt concentration increased, the portion of
substrate digested to smaller fragments decreased. At 0 mM
added NaCl, the 18-nt PCP was extensively digested into several smaller
fragments. By 100 mM NaCl, it was predominantly digested by
one nucleotide; by 150 mM, significant inhibition was
observed; and by 200 mM NaCl, the exonuclease activity was
almost completely inhibited. By contrast, the AP activity appeared
relatively insensitive to the NaCl concentration. The progression of
the exonuclease on the substrate was enhanced by the presence of excess
zinc, indicating that zinc may be important for processivity of the
Endo IV on its substrate in addition to its known role in
phosphodiester bond cleavage (22). By contrast, magnesium and manganese
appeared to inhibit progression of the exonuclease activity on the
substrate (although MnSO4 appeared to have no effect). EDTA
inhibited progression of the exonuclease activity and also had an
inhibitory effect on the AP activity. The addition of the reducing
agent DTT inhibited the progression of the enzyme on the substrate.
This was an unexpected finding and suggests that the exonuclease
activity of the enzyme may be more active when the cell is under
oxidative stress. The Endo IV exonuclease activity may be processive or
distributive, and further experiments are needed to clarify this issue.
Typical Endo IV assays in previous reports contained 100 mM
KCl, 1 mM DTT, and 1 mM EDTA in the reaction
mixture (10, 11). The effect of cumulative addition of the agents to
the reaction mixture on the Endo IV exonuclease activity was assessed.
Addition of any of the agents reduced progress of the enzyme on the
substrate, and addition of all three agents completely inhibited the
exonuclease activity. Because previous characterizations of the Endo IV
enzyme have predominantly used buffers including all three agents, this may explain why the exonuclease activity was not observed previously.
The results shown indicate that the Endo IV exonuclease activity works
very well within an AP site cleavage context and has a strong
preference for substrates with either 3' recessed ends or an incised AP
site (Fig. 8) and raises the question as to what the extent of
exonuclease activity in vivo is at such sites.
Reconstitution of the E. coli base excision-repair pathway
using crude or purified enzymes, including Endo IV showed no detectable
resynthesis of DNA 5' of AP sites (5). However, the conditions used are
likely to have been refractory to Endo IV exonuclease activity.
Comparison of the relative AP endonuclease and exonuclease activity of
Endo IV indicates that the exonuclease activity is high and that
exonuclease action is likely to follow AP incision and have a
functional significance in vivo. Although the function of
the 3'-5' exonuclease activity of Exo III is unknown and has been
considered redundant, the discovery of substantial exonuclease activity
associated with Endo IV argues in favor of a significant functional
importance for this activity in vivo.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Bruce Demple for
providing E. coli Endo IV and for helpful suggestions and to
Dr. Pat Vaughan (HiberGen) for carrying out the mass spectrometry
studies and for helpful comments.
 |
FOOTNOTES |
*
This work was supported by the Higher Education Authority
under the program for research in third level institutions.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. E-mail:
t.mccarthy@ucc.ie.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M210750200
 |
ABBREVIATIONS |
The abbreviations used are:
AP, apurinic and
apyrimidinic;
Exo, exonuclease;
Endo, endonuclease;
Ec-Endo IV, E. coli Endo IV gene;
Tm-Endo IV, T. maritima
Endo IV gene;
MES, 4-morpholineethanesulfonic acid;
DTT, dithiothreitol;
PCP, primary cleavage product;
nt, nucleotide(s).
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.