From the Department of Pharmacological Sciences, State University
of New York at Stony Brook, Stony Brook, New York 11794-8651
Apurinic/apyrimidinic (AP) sites occur frequently
in DNA as a result of spontaneous base loss or following removal of a
damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5'-deoxyribose phosphate (dRP)
residue that must be removed during the base excision repair process.
This 5'-dRP group may be removed by AP lyase enzymes that employ a
-elimination mechanism. This
-elimination reaction typically
involves a transient Schiff base intermediate that can react with
sodium borohydride to trap the DNA-enzyme complex. With the use of this
assay as well as direct 5'-dRP group release assays, we show that T4
DNA ligase, a representative ATP-dependent DNA ligase,
contains AP lyase activity. The AP lyase activity of T4 DNA ligase is
inhibited in the presence of ATP, suggesting that the adenylated lysine
residue is part of the active site for both the ligase and lyase
activities. A model is proposed whereby the AP lyase activity of DNA
ligase may contribute to the repair of abasic sites in DNA.
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INTRODUCTION |
DNA repair pathways have evolved to process a wide range of
chemically distinct lesions in DNA (1). One of the most common types of
damage is spontaneous or enzymatic hydrolysis of the N-glycosidic bond between a DNA base and the sugar phosphate
backbone generating an abasic site (AP
site).1 AP sites are highly
mutagenic and require rapid and efficient repair. AP sites are
processed by a base excision repair pathway that is frequently
initiated by the action of a class II AP endonuclease that cleaves the
DNA backbone adjacent to the lesion to produce 3'-OH and 2'-deoxyribose
5'-phosphate termini (2). The latter residue, referred to as a 5'-dRP
moiety, is relatively alkali labile and can be removed by AP lyases
that facilitate
-elimination. Most enzymes demonstrated to have
dRPase activity operate through this lyase mechanism, although some,
such as the Escherichia coli recJ protein, catalyze
hydrolysis (3). The two classes of dRPase enzymes can be distinguished
by the fact that the lyase mechanism frequently involves formation of a
transient Schiff base intermediate in which an amino group on the
enzyme is covalently bound to the DNA. This lyase mechanism, first
proposed for E. coli endonuclease III (4), provides a simple
method to identify a polypeptide with AP lyase activity because the
Schiff base intermediate can be trapped in a stable form by reaction
with a strong reducing agent, such as NaBH4 or
NaBH3CN. Thus, with an appropriate radioactively labeled
substrate, the label can be transferred to the AP lyase enzyme. This
borohydride trapping has been documented for several repair enzymes
(5-9) and, more recently, for DNA pol
(10, 11).
The combined action of AP endonuclease and AP lyase leaves a
one-nucleotide gap that is filled by a DNA polymerase. The final step
in repair involves DNA strand sealing by DNA ligase. The mechanism of
DNA ligase involves covalent modification of the enzyme by adenylation,
transfer of the AMP residue in a phosphoanhydride linkage to the
5'-phosphate of nicked DNA, followed by resealing of the DNA strand
driven by the energy of AMP hydrolysis.
We recently characterized a mtDNA ligase as part of an effort to
reconstitute repair of AP sites using mitochondrial enzymes (12). The
size, template specificity, and immunological properties of the mtDNA
ligase suggested that this was a form of DNA ligase III. In the course
of this work we found that mtDNA ligase is active on a DNA substrate
containing an AP site incised on the 5' side by a class II AP
endonuclease. Our observations prompted a detailed investigation of the
action of T4 DNA ligase as a prototype for ATP-dependent
DNA ligases at AP sites in DNA. In this paper we show that in the
presence of ATP, T4 DNA ligase is able to reseal an incised AP site. In
the absence of ATP, T4 DNA ligase acts as an AP lyase to facilitate a
-elimination reaction that leads to removal of the 5'-dRP residue. A
model is presented whereby an intrinsic AP lyase activity in DNA ligase
may facilitate repair of AP sites.
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EXPERIMENTAL PROCEDURES |
Materials--
mtDNA ligase, mitochondrial AP endonuclease, and
DNA ligase I were purified from Xenopus ovary tissue as
described (12). T4 DNA ligase was obtained from Boehringer Mannheim. T7
DNA ligase was a gift from Dr. J. Dunn (Brookhaven National
Laboratory). Variable quantities of both preparations of bacteriophage
DNA ligase were subjected to SDS-PAGE analysis (13) in parallel with
standard proteins of known concentration. The gels were stained with
Coomassie Blue to confirm that both preparations were essentially homogeneous and to permit estimation of protein concentrations using
densitometry of the stained gels. Radiochemicals were purchased from
ICN Radiochemicals. Uracil DNA glycosylase (UDG) was obtained from
Epicentre Technologies (HK-UNG). FPG protein was a gift from J. Tchou
and A. P. Grollman (SUNY-Stony Brook). Sodium borohydride and
sodium thioglycolate were obtained from Sigma-Aldrich. Other reagent
grade chemicals were obtained from Sigma-Aldrich or Fisher. The Poros Q
4.6 × 50-mm column used for anion exchange HPLC was obtained from
Perceptive Biosystems. Oligonucleotides were either synthesized by the
phosphoramidite method at the SUNY-Stony Brook Oligonucleotide
Synthesis Facility or were obtained from Operon. A continuous duplex
oligonucleotide was prepared by annealing a 5'-32P
kinase-labeled 32-mer (5'-CATGGGCCGACATGAUCAAGCTTGAGGCCAAG) to a
complementary oligonucleotide (5'-TCTTGGCCTCAAGCTTGATCATGTCGGCCCATG). Two nicked duplex oligonucleotides were prepared by annealing a
5'-32P kinase-labeled 17-mer (5'-UCAAGCTTGAGGCCAAG;
referred to as U17) and either a nonradioactive 15-mer
(5'-CATGGGCCGACATGA) or 12-mer (5'-GGGCCGACATGA) to the same
complementary strand described above. The 12-mer was used in the
experiment in Fig. 7C to permit better resolution of the
reaction product from the initial labeled substrate.
Methods--
Oligonucleotides were phosphorylated using standard
procedures (14) and annealed by heating to 80 °C in 0.1 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA
and slow cooling to 4 °C. Oligonucleotides were treated immediately
before use with UDG in 20 mM Hepes, pH 7.5, and diluted
into an assay mixture with DNA ligase in 10 mM Hepes, pH
7.5, 1 mM MgCl2, unless otherwise indicated.
Borohydride trapping was performed by a modification of published
procedures (9, 11) by including 20 or 50 mM
NaBH4 in the binding reaction. After 30 min at 25 °C,
the solution was adjusted to contain 6 mM CaCl2
and 10 µg/ml micrococcal nuclease. After 20 min of incubation at
37 °C, proteins were precipitated with trichloroacetic acid, analyzed by SDS-polyacrylamide gel electrophoresis (13), and detected
by autoradiography or PhosphorImager analysis (Molecular Dynamics).
dRPase activity was measured as described (3), and HPLC analysis of
products generated in the presence of sodium thioglycolate was
performed as described (5, 15), except that a Poros Q anion exchange
column was used.
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RESULTS AND DISCUSSION |
DNA Ligases Are Able to Reseal DNA Strands Nicked on the 5' Side of
an AP Site--
Our laboratory has studied the base excision repair
pathway with nuclear and mitochondrial protein fractions using
templates containing precisely positioned single AP sites embedded in
covalently closed circular DNA (12, 15). These sites are readily
cleaved on the 5' side by class II AP endonuclease to yield a 3'-OH
terminus and a 5'-deoxyribose phosphate (dRP) residue. When templates
bearing incised AP sites are incubated with DNA ligase in the presence of ATP, the DNA ligases are able to reseal the nicked strand (Fig. 1). This reaction has been reported for
T4 DNA ligase (16) but not for eukaryotic DNA ligases. Because ligation
of a 5'-dRP moiety directly reverses the action of AP endonuclease, it
is counterproductive for repair. It is also a potential confounding
factor in efforts to reconstitute repair reactions in vitro,
because religation of an abasic site might not be differentiated from
actual repair. In the experiment in Fig. 1, we used a substrate with a
synthetic analogue of an abasic site, a tetrahydrofuran analogue that
has been used extensively in repair studies (15, 17). This is an
analogue of a reduced deoxyribose moiety and is not subject to
-elimination. Experiments presented below show that T4 DNA ligase
can also reseal an authentic (nonreduced) AP site.

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Fig. 1.
DNA ligases can reseal a nick adjacent to a
5'-phosphorylated abasic site. A 5'-end labeled oligonucleotide
containing a single synthetic AP site
(3-hydroxy-2-hydroxymethyltetrahydrofuran, designated F for furan) was
ligated into a gapped heteroduplex, and the covalently closed circular
DNA product was purified as described (15). PAGE-urea gel analysis of a
HinfI digest of this substrate confirmed that all substrate
molecules were ligated with the tetrahydrofuran residue embedded in a
46-mer fragment, denoted as 46(F) (lane 1).
Treatment with mitochondrial AP endonuclease led to cleavage on the 5'
side of the tetrahydrofuran residue, providing a 26-mer fragment
following diagnostic HinfI cleavage (lane 2).
This fragment with a 5'-tetrahydrofuran residue is identified as
F26. Samples of this mitochondrial AP endonuclease-incised
substrate were incubated with X. laevis DNA ligase I, mtDNA
ligase, T4 DNA ligase, or T7 DNA ligase (lanes 3-6). The
products were deproteinized by organic extraction and cleaved with
HinfI endonuclease prior to electrophoresis. The radioactive
species moving slightly slower than the F26 fragment, which is most
apparent in lane 6, is an intermediate in the ligase
reaction produced by transfer of AMP to the 5' terminus at the
nick.
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T4 DNA Ligase Has AP Lyase Activity--
We previously showed
Xenopus laevis mtDNA ligase can be labeled using a
borohydride trapping procedure that is specific for AP lyase activities
(12). To determine whether this is a general property of
ATP-dependent DNA ligases, we tested T4 and T7 DNA ligases
for the ability to react with AP sites in a borohydride trapping assay.
This assay employed an oligonucleotide substrate designed to contain a
specific U residue adjacent to a nick, referred to as 15-*U17:33 to
denote a 15-mer annealed adjacent to a kinase-labeled (*) 17-mer to a
33-mer complementary strand. Treatment of the duplex oligonucleotide
with UDG results in a 5'-phosphoryl abasic site. This is an exact model
of the substrate that would be generated by action of a class II AP
endonuclease. Fig. 2 shows that both T4
DNA ligase and T7 DNA ligase react in this assay. Controls in
lanes 3 and 4 of Fig. 2 demonstrate that
cross-linking was dependent on the presence of NaBH4 and on
the AP site, because the reaction was not observed with a control
oligonucleotide containing thymine in place of uracil.

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Fig. 2.
Bacteriophage DNA ligases react in a
borohydride trapping assay for AP lyases. An autoradiogram of an
SDS-PAGE analysis of labeled DNA ligases is shown with the positions of
protein molecular mass markers in kDa indicated on the left.
To provide radioactively labeled markers, T4 and T7 DNA ligase were
incubated with [ -32P]ATP (lanes 1) in a
buffer containing 20 mM Tris, pH 8.0, 10 mM
MgCl2, 5 mM dithiothreitol, 0.02% Triton X-100
to permit adenylation of the active site lysine. For borohydride
trapping reactions, DNA ligase was incubated with a duplex
oligonucleotide with an internal nick adjacent to a
5'-32P-U residue (lanes 2 and 3) or
with a 5'-32P-T residue as a control (lanes 4)
as diagramed at the bottom of the figure. 1 pmol of the
oligonucleotide substrate was incubated with 0.1 unit of UDG and 100 ng
of either T4 DNA ligase or T7 DNA ligase in 20 µl of 10 mM Hepes, pH 7.5, 1 mM MgCl2 and
either 50 mM NaBH4 (lanes 2 and
4) or 50 mM NaCl (lanes 3). The
reactions were treated with micrococcal nuclease and labeled proteins
were detected by following SDS-PAGE as described under "Experimental
Procedures."
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We performed a variety of control experiments to characterize the
putative AP lyase activity in T4 DNA ligase. The extent of
cross-linking with a nicked substrate varies with solution conditions.
Cross-linking is reduced at 5 mM MgCl2 in
comparison with the standard reaction conducted at 1 mM
MgCl2 and is inhibited more than 90% by 1 mM
ATP (Fig. 3). The standard reaction
includes post-treatment with micrococcal nuclease to degrade the
oligonucleotide cross-linked to protein by NaBH4. When this
nuclease treatment was omitted, the electrophoretic mobility of the
major cross-linked protein species was reduced, as expected for a
DNA-protein complex (Fig. 3B). In the absence of micrococcal
nuclease treatment a minor cross-linked labeled species with
essentially the same gel mobility as unmodified T4 DNA ligase was also
observed. This faster migrating species is expected to be formed as an
intermediate in the action of an AP lyase, as shown in Fig.
4. The initial product of an attack by AP
lyase on an AP site is a Schiff base intermediate in which the enzyme
is joined to DNA. The AP lyase reaction proceeds with elimination of
the DNA from the C3' position of deoxyribose, producing an enzyme-dRP
intermediate with a Schiff base linkage. Both the enzyme-DNA and
enzyme-dRP species can be reduced by NaBH4 to generate the
doublet of cross-linked species in Fig. 3B. These
borohydride cross-linking results are consistent with the hypothesis
that T4 DNA ligase contains AP lyase activity.

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Fig. 3.
Characterization of the borohydride trapping
reaction for T4 DNA ligase. A, the UDG-treated nicked
oligonucleotide substrate used in Fig. 2 was incubated with T4 DNA
ligase in reactions containing 10 mM Hepes, pH 7.5, 50 mM NaBH4 and the indicated concentrations (in
mM) of either MgCl2 or EDTA and with ATP in one
reaction (lane 5). B, standard borohydride
trapping reactions with T4 DNA ligase were performed, comparable with
lane 2 of A. Lane 2 shows the effect
of omitting post-treatment with micrococcal nuclease (MN).
In both panels, the arrowheads indicate the mobility of T4
DNA ligase.
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Fig. 4.
The chemistry of AP lyase action accounts for
DNA-enzyme and dRP-enzyme complexes following NaBH4
treatment. This scheme is based on those presented for other AP
lyase enzymes (7, 19). The Shiff base reaction scheme requires attack
by a free amino group of the AP lyase on the C1' residue of the
deoxyribose. This reactive nitrogen is referred to as N-enz.
Other functional groups within the enzyme may assist in the
-elimination reaction as indicated. Covalent intermediates in the
Schiff base reaction scheme labeled 1 and 2 may
be reduced by borohydride to yield stable species with the protein
cross-linked to an oligonucleotide or to a dRP moiety,
respectively.
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In other experiments, we found that it was not necessary to present the
AP site in the context of a nick in DNA, although this is the preferred
substrate. Borohydride trapping was observed when the 15-mer
oligonucleotide was omitted from the standard nicked substrate, leaving
a 5'-AP site adjacent to single-stranded DNA. We also observed
cross-linking to a free oligonucleotide with a 5'-dRP site generated by
the action of UDG on the 5'U-17-mer oligonucleotide
(5'-32P-UCAAGCTTGAGGCCAAG). The efficiency of borohydride
trapping with a single-stranded 5'-AP oligonucleotide was about 50% of
that observed with the nicked oligonucleotide substrate. The
single-stranded oligonucleotide substrate was used in some experiments
described below to simplify preparation of large quantities of
substrate for AP lyase assays.
The borohydride trapping reaction is a very sensitive probe of AP lyase
activity but is not always efficient because it acts on a transient
intermediate in the overall AP lyase reaction. For DNA glycosylases
that remove a base and attack the AP site in a sequential dual
mechanism, such as FPG protein, this borohydride trapping procedure is
relatively easy to control. It is more difficult to trap a large
fraction of a protein that does not contain an intrinsic glycosylase
activity because the NaBH4 required to cross-link the
enzyme-DNA Schiff base intermediate can also react with the ring open
form of the sugar residue to inactivate the substrate. As an
independent assay for AP lyase activity, we monitored the release of
free 5'-dRP from DNA as an acid or ethanol-soluble species, as shown in
Fig. 5. When reactions were performed
with a high concentration of the single stranded 5'-dRP-17-mer
oligonucleotide, we found that dRP release was linear with time, showed
a clear temperature optimum, and was inhibited by ATP as observed for the borohydride trapping assay. The ethanol-soluble species released by
T4 DNA ligase in the presence of thioglycolic acid was observed to have
the same chromatographic properties as the product released by FPG
protein, which has a well characterized AP lyase activity (Fig.
6 and Refs. 5, 7, and 18).

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Fig. 5.
Release of an acid soluble product from a
5'-32P-labeled abasic site by T4 DNA ligase. 15 pmol
of 5'-32P U17 oligonucleotide pretreated with UDG was
incubated with 100 ng of T4 DNA ligase in 40 mM Hepes
buffer, pH 7.5, for varied periods of time at 30 °C (A),
for 30 min at varied temperature (B), or in the presence of
increasing concentrations of ATP (C). dRP release was
measured as the generation of a radioactive product soluble in the
presence of cold TCA and activated charcoal (3). The percentage of dRP
removed was determined relative to the total alkali-labile cpm. The
maximal amount of label solubilized in parallel reactions without
enzyme represented 4% of the total available substrate.
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Fig. 6.
The putative 5'-dRP product released by DNA
ligase has the same chromatographic properties as that released by FPG
protein, a well characterized AP lyase. The 5'-32P U17
oligo pretreated with UDG was incubated with either FPG protein as a
positive control (A) or with T4 DNA ligase (B) in
the presence of 50 mM sodium thioglycolate. The product
released by the lyase activity of FPG protein has been shown to react
with thioglycolate to generate an anionic species (5, 18). Reaction of
the -elimination product with thioglycolate leads to reduction of
the aldehyde, blocking the subsequent -elimination reaction for FPG
protein. The ethanol-soluble reaction products were analyzed by
chromatography on a Poros Q HPLC column using the indicated NaCl
gradient. The intact oligonucleotide elutes from this column with the 1 M NaCl step.
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The borohydride trapping and dRP release assays show that T4 DNA ligase
is an authentic AP lyase by the same criteria used to show that DNA pol
possesses AP lyase activity (10, 11). Although T4 DNA ligase is
capable of processing multiple AP site substrates (Fig. 5A),
the turnover number is less than 10% of the vigorous rate observed for
FPG protein (5). It should be noted that the glycosylase activity of
FPG protein is reported to be significantly slower than the AP lyase
activity (5). A lower turnover number for the AP lyase activity in T4
DNA ligase may be expected because the enzyme is likely to bind
persistently to the gapped substrate generated by AP lyase action on a
site previously cleaved by AP endonuclease.
We have not yet identified the active site for the DNA
ligase-associated AP lyase. The borohydride trapping reaction requires attack on the C1' residue of deoxyribose by an N-terminal amino group
or by an internal lysine (19). The fact that the AP lyase activity is
suppressed by ATP suggests that the active site lysine residue that is
adenylated in DNA ligase (20) may be involved directly in the
nucleophilic attack that promotes
-elimination. This residue is
normally in close proximity to the nick in a DNA substrate in the
course of a DNA ligation reaction. However, we have not ruled out the
possibility that another lysine residue may be involved in this attack,
because the deadenylated enzyme may have an altered conformation that
interacts differently with DNA substrates containing 5'-dRP residues.
It will be particularly interesting to map the active site residue in
T7 DNA ligase that reacts in the borohydride trapping reaction because
the structure of this enzyme has been determined (21). This enzyme has
the added advantage that it is relatively small, with only 359 amino acid residues.
A Model for the Role of AP Lyase Associated with DNA
Ligase--
We have observed AP lyase activity using the borohydride
trapping assay for the following four different
ATP-dependent DNA ligases: T4 and T7 DNA ligase (Fig. 2),
mtDNA ligase (12), and DNA ligase I (data not shown). Because
ATP-dependent DNA ligases as a class share structural and
functional features (22), it is likely that the presence of AP lyase
activity will be conserved in this family. To date we have not been
able to document AP lyase activity in bacterial DNA ligases from either
E. coli or T. aquaticus either in the presence or
the absence of their cofactor, NAD (data not shown).
The critical question raised by our observations is whether the AP
lyase activity associated with T4 DNA ligase plays a significant physiological role. A model for the action of T4 DNA ligase at AP sites
is shown in Fig. 7. In living cells, AP
sites are very rapidly incised by AP endonuclease to generate the sort
of nicked AP substrate we have used in our reactions. The experiments
reported here show that T4 DNA ligase can act as an AP lyase at these
sites in the absence of ATP. Under these conditions, the deadenylated enzyme cannot seal the nick and instead facilitates
-elimination, leading to loss of the 5'-dRP residue. This produces the single nucleotide gap structure diagramed as species 5 in Fig. 7. This single
base gap may be repaired by the action of DNA polymerase and the
conventional strand sealing action of DNA ligase.

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Fig. 7.
Model for the action of DNA ligase at AP
sites. A, the proposed action of DNA ligase at an incised AP
site is illustrated in the presence or the absence of ATP. DNA ligase
is shown as a C-shaped enzyme with a prominent groove (21). See
"Results and Discussion" for details. B, a substrate
containing an internal AP site was prepared by UDG treatment of a
duplex oligonucleotide with a single U residue 15 nucleotides from the
32P-labeled 5'-end as diagramed at the bottom.
The oligonucleotide was incubated for 45 min at 25 °C in 40 mM Hepes, pH 7.5, either without additional enzyme
(lane 1) or with either E. coli FPG protein (lane
2) or T4 DNA ligase (lane 3). Reactions were treated for 15 min with 0.1 M NaBH4 to reduce the abasic sugar
residue, and products were subjected to electrophoresis on a 20%
PAGE-urea gel and detected by autoradiography. C, an
oligonucleotide substrate was prepared by annealing a nonradioactive
12-mer adjacent to the 5'-labeled U17 oligomer to a complementary
strand as diagramed at the bottom. This duplex was treated
with UDG to create a 5'-dRP residue and then incubated in 40 mM Hepes, pH 7.5, buffer alone (lane 1) or with
20 ng of T4 DNA ligase without ATP (lane 2) or with the same
amount of T4 DNA ligase with 0.1 mM ATP (lane
3). All incubations were for 1 h at 25 °C. Reactions were
treated for 15 min with 0.1 M NaBH4, and
products were subjected to electrophoresis on a 20% PAGE-urea gel and
detected by autoradiography. Markers consisted of kinase-labeled
oligonucleotides identified by size on the left. The 17-mer
marker is the intact U17 oligonucleotide, and the 12-mer marker is a
5'-labeled sample of the same nonradioactive 12-mer used to prepare the
substrate. The diagrams at the bottom of B and
C show the oligonucleotides used in these experiments with
the top strands oriented 5' to 3'.
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It is also important to consider the action of T4 DNA ligase at
incised AP sites in the presence of ATP, because a large fraction of
DNA ligase may exist in the adenylated state in vivo. Our
results suggest that the adenylated T4 DNA ligase has a reduced ability to promote
-elimination. Instead, when a T4 DNA ligase molecule that
is activated by adenylation binds this nicked substrate, it seals the
nick to regenerate an internal AP site, as shown in Fig. 1 and
diagramed as species 3 in Fig. 7A. The second product of the
ligation reaction is a "disarmed" DNA ligase molecule that is no
longer adenylated but is still in contact with the AP site. To test
whether T4 DNA ligase is able to incise DNA on the 3' side of an
internal AP site (i.e., without prior action of an AP
endonuclease), we performed the experiment in Fig. 7B. This experiment shows that T4 DNA ligase is clearly capable of strand incision to yield a product with a slightly slower gel mobility than
that produced by the well characterized AP lyase of FPG protein. This
suggests that T4 DNA ligase is able to promote
-elimination but
unlike FPG protein does not efficiently promote
-elimination. This
sort of incision reaction was not observed in Fig. 1 because that
experiment employed a reduced AP site analogue. These results suggest
that when T4 DNA ligase seals a nick generated by class II AP
endonuclease, it may recognize the product as a mistake and employ its
lyase activity to reopen the DNA. To test this prediction, we performed
the experiment shown in Fig. 7C. In this experiment, T4 DNA
ligase was incubated with a 17-mer oligonucleotide containing a
5'-32P-dRP residue adjacent to a nonradioactive 12-mer. DNA
ligase was able to ligate the 12-mer to the 5'-dRP-17 mer to generate a
29-mer with an internal 32P-dRP residue (lane 3 of Fig. 7C). A limited extent of ligation was observed
without the addition of exogenous ATP (lane 2), presumably because a fraction of the T4 DNA ligase is purified in an adenylated form. The more efficient ligation in the presence of ATP was followed by incision on the 3' side of the AP site to produce a labeled 12-mer
with a 3'-dRP residue. Thus, the label transfer experiment in Fig.
7C confirms the model for the action of T4 DNA ligase at an
AP site in the presence of ATP. The ring open 3'-dRP residue produced
by AP lyase cannot be rejoined by DNA ligase due to the 2'-3'-double
bond, but the 3'-dRP group would be susceptible to release by class II
AP endonuclease. Taken together, these experiments suggest that the
role of T4 DNA ligase in base excision repair may not be limited to the
final step of strand closure.
We thank J. Dunn, F. Johnson, Y. Matsumoto,
and B. Weiss for helpful discussion and for comments on the manuscript
and J. Tchou and J. Dunn for gifts of E. coli FPG protein
and T7 DNA ligase, respectively.