From the Institute of Veterinary Biochemistry, University of
Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
The joining of single-stranded breaks in
double-stranded DNA is an essential step in many important processes
such as DNA replication, DNA repair, and genetic recombination. Several
data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA
polymerases
and
show multiple functions in different DNA
transactions, we investigated the effect of DNA ligase I on various DNA
synthesis events catalyzed by these two essential DNA polymerases. DNA
ligase I inhibited replication factor C-independent DNA synthesis by
polymerase
. Our results suggest that the inhibition may be due to
DNA ligase I interaction with proliferating cell nuclear antigen (PCNA)
and not to a direct interaction with the DNA polymerase
itself.
Strand displacement activity by DNA polymerase
was also affected by
DNA ligase I. The DNA polymerase
holoenzyme (composed of DNA
polymerase
, PCNA, and replication factor C) was inhibited in the
same way as the DNA polymerase
core, strengthening the hypothesis
of a PCNA interaction. Contrary to DNA polymerase
, DNA synthesis by
DNA polymerase
was stimulated by DNA ligase I in a
PCNA-dependent manner. We conclude that DNA ligase I
displays different influences on the two multipotent DNA polymerases
and
through PCNA. This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.
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INTRODUCTION |
DNA ligases play essential roles in important cellular pathways,
such as DNA replication, DNA recombination, and DNA repair, by joining
single- and double-stranded breaks in an ATP-dependent manner (1). Four DNA ligases (I, II, III, and IV), the functions of
which are not yet completely understood, have been identified in
mammalian cells (2). Human DNA ligase I is a monomer of 102 kDa (3)
composed of two clearly distinct regions as follows: a highly conserved
78-kDa C-terminal domain containing the active site (4), and a 24-kDa
N-terminal region that is not required for ligase activity but contains
the nuclear localization signal and directs the enzyme to sites of DNA
replication (5). Several of the following observations indicate an
involvement of DNA ligase I in DNA replication: (i) DNA ligase I is
responsible for a major part of DNA ligase activity in proliferating
mammalian cells (6-9); (ii) cytostaining experiments with antibodies
against DNA ligase I showed that the enzyme co-localizes in the nucleus
with DNA polymerase (pol)1
(10); (iii) the enzyme co-purifies with a protein complex competent
in in vitro SV40 DNA replication (11); (iv) a mutation in
the DNA ligase I gene in the human 46BR cell line leads to a delay in
the joining of the Okazaki fragments (12). These, together with several
other observations (3, 13), imply an important role of DNA ligase I in
DNA replication as well as in DNA repair (5, 14). Experiments by
Mackenney et al. (15) suggest that DNA ligase I, through its
N-terminal region, interacts with other proteins.
The most important proteins in the DNA synthesis reaction are the pols.
So far six pols have been identified in eukaryotic cells, called
,
,
,
,
, and
(reviewed in Refs. 16 and 17). Three of
them (
,
, and
) have been shown to be essential in DNA
replication (18-20). pol
is a complex consisting of four polypeptides as follows: a 180-kDa subunit that harbors the polymerase activity, a 70-kDa peptide of uncertain function, and two small subunits of 48 and 58 kDa, respectively, containing the primase activity. pol
is responsible for initiation of DNA replication; the
primase synthesizes RNA primers, which are then elongated into DNA
primers by the polymerase activity on both leading and lagging strands
(21). It is moderately processive and dissociates from the DNA,
facilitating a switch to a highly processive, proofreading pol such as
pol
. pol
consists of at least two identified subunits with
molecular masses of 125 and 50 kDa. The polymerase and the 3'
5'
exonuclease activities are both located on the large subunit (22, 23).
pol
exhibits very low activity on its own, but upon addition of the
auxiliary factor proliferating cell nuclear antigen (PCNA) (reviewed in
Refs. 24 and 25), the activity and the processivity are stimulated up
to 100-fold (26, 27). PCNA is loaded onto DNA by a second auxiliary
factor, replication factor C (RF-C) (reviewed in Ref. 28), in an
ATP-dependent fashion. RF-C recognizes the 3'-end of the
nascent strand, thereupon inducing the dissociation of pol
/primase
and recruiting pol
(29), which then performs processive leading
strand synthesis. On the lagging strand, pol
has been shown
in vitro to extend the Okazaki fragments (13, 21). Whether
this is also the case in vivo remains controversial, since
pol
has been proposed as the lagging strand pol (30-33). pol
is only not implicated in DNA replication but also plays an important
role in DNA repair (reviewed in Ref. 17) and V(D)J recombination (34).
pol
is enzymatically distinguishable from pol
by its differing
response to PCNA. Whereas pol
is processive only in the presence of
PCNA, pol
is very active even in the absence of PCNA. Previous
studies have reported that pol
is completely unresponsive to the
addition of PCNA (20, 35); however, more detailed kinetic studies have
shown that PCNA can also stimulate the processivity of pol
and that
pol
interacts with PCNA, increasing its rate of nucleotide
incorporation (36). In the presence of RF-C and ATP, pol
, like pol
, is able to form a stable complex with PCNA at the 3'-end of the
primer, called pol
holoenzyme (30, 37). Similar to pol
, the pol
also has a role in DNA repair (38, 39), and a function in the
repair of double-stranded DNA breaks has been suggested (40).
In this report we show that pols
and
are affected in different
ways by DNA ligase I. DNA elongation by pol
is strongly inhibited
by DNA ligase I, most likely through the already observed interaction
of DNA ligase I with PCNA (41). DNA synthesis by pol
on the other
hand is stimulated by DNA ligase I. Here PCNA plays a dual role; at low
concentrations pol
stimulation by DNA ligase I is increased,
whereas at high concentrations the stimulatory effect is canceled. All
these effects are ATP-independent, that is they do not involve the DNA
ligase I per se and consequently appear to be caused by
protein-protein interactions between PCNA and DNA ligase I.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Radiolabeled nucleoside triphosphates were
purchased from Amersham Pharmacia Biotech, and unlabeled dNTPs were
from Boehringer Mannheim. All other reagents were of analytical grade
and were purchased from Merck (Darmstadt, Germany) or Fluka (Buchs,
Switzerland).
Nucleic Acids--
The single-stranded M13 DNA was hybridized to
the universal sequencing primer as outlined (42). The homopolymer
poly(dA)200 (Amersham Pharmacia Biotech) was mixed in the
desired weight ratio with the oligomer oligo(dT)12-18
(Amersham Pharmacia Biotech) in 20 mM Tris-HCl (pH 8.0),
containing 20 mM KCl and 1 mM EDTA, heated at 60 °C for 5 min with subsequent slow cooling to room temperature. Gapped double-stranded DNA was constructed as described (43).
Enzymes and Proteins--
Recombinant human DNA ligase I was
purified (15). Human PCNA was overexpressed in Escherichia
coli BL21(DE3) harboring the expression plasmid pT7/PCNA as
described (44). The phosphorylated PCNA was expressed in E. coli, purified, and phosphorylated in vitro using
[
-32P]ATP as described (45). Recombinant RF-C was
purified from baculovirus infected HighFive cells (46). Pols
and
were purified from fetal calf thymus as described (47). One unit of
enzyme activity corresponds to the incorporation of 1 nmol of
total dTMP into acid-precipitable material in 60 min at 37 °C in a standard assay containing 500 ng of
poly(dA)/oligo(dT)10:1 and 20 µM dTTP.
RF-C-independent pol
Assay--
A final volume of 25 µl
contained the following: 50 mM BisTris-HCl (pH 6.5), 6 mM MgCl2, 1 mM DTT, 250 µg/ml
BSA, 20 µM [3H]dTTPs (400 cpm/pmol), 500 ng
of poly(dA)/oligo(dT), 120 ng of PCNA (unless differently mentioned)
and pol
to be titrated. The reactions were incubated at 37 °C
for the indicated times and precipitated with 10% trichloroacetic
acid, and the insoluble radioactive material was determined as
described (48).
RF-C-independent pol
Assay--
A final volume of 25 µl
contained the following: 75 mM Hepes-NaOH (pH 7.5), 1 mM DTT, 20% (v/v) glycerol, 250 µg/ml BSA, 10 mM MgCl2, 10 mM KCl, 20 µM [3H] dTTPs (400 cpm/pmol), 500 ng of
poly(dA)/oligo(dT), and pol
to be titrated. Reactions were analyzed
as described above for pol
.
RF-C-dependent pol
/
Assay--
A final volume
of 25 µl contained the following: 40 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 1 mM ATP, 5 mM DTT, 200 µg/ml BSA, 15 µM
[3H]dNTPs (300 cpm/pmol), 100 ng of singly primed M13
DNA, 120 ng of PCNA, 350 ng of single-stranded DNA-binding protein, 20 ng of RF-C, and 0.25 units of pol
(or 0.16 units pol
). The
reactions were incubated for 60 min at 37 °C, stopped, and
quantified as described (48).
RF-C-dependent PCNA Loading--
A recombinant PCNA
form that could be artificially phosphorylated in vitro at
the N terminus by cAMP-dependent protein kinase was used
(45). A final volume of 25 µl contained the following: 40 mM triethanolamine HCl (pH 7.5), 200 µg/ml BSA, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 50 ng of 32P-phosphorylated PCNA, 50 ng
of RF-C, 60 ng of gapped circular DNA (49), and DNA ligase I in the
amount indicated in the figure. The samples were incubated for 3 min at
37 °C, followed by the addition of glutaraldehyde to a final
concentration of 0.1% (w/v) and further incubation at 37 °C for 10 min. The samples were then adjusted with 2.5% Ficoll-400 and marker
dyes, loaded on a 0.8% agarose gel, and electrophoresed in 45 mM Tris borate buffer (pH 8.3) containing 1 mM
EDTA and 0.1% SDS. The gel was fixed in 10% acetic acid, 12%
methanol, dried, and exposed to x-ray film. PCNA loading onto DNA
was quantified by PhosphorImager (Molecular Dynamics).
Strand Displacement DNA Synthesis--
The steps of the DNA
substrate synthesis are described (see Ref. 43). Gapped dsDNA was first
digested with KpnI and PvuII in suitable buffer
conditions for 1 h at 37 °C. Digested DNA was used for the
experiment without further purification. For strand displacement DNA
synthesis the following components were mixed in a final volume of 25 µl: 50 mM BisTris (pH 6.5), 6 mM
MgCl2, 1 mM DTT, 250 µg/ml BSA, 120 ng of
PCNA, 5 µCi of [
-32P]dATP (3000 Ci/mmol), 20 ng of
gapped dsDNA, and 0.25 units pol
. The mixture was incubated for 7 min at 37 °C to allow incorporation of the first two dATPs. Then
dCTP, dGTP, and dTTP (30 µM each) and DNA ligase I (in
amounts as described in the figure legend) were added. DNA synthesis
was allowed to continue for 30 min at 37 °C. Reactions were
terminated by heating for 10 min at 70 °C. After cooling to room
temperature, reactions were treated with proteinase K (60 µg/ml), in
the presence of 1% SDS (w/v) and 20 mM EDTA (pH 8.0) for
30 min at 37 °C. After addition of the same volume of 100%
formamide and marker dyes, samples were loaded on a 8% polyacrylamide
gel, containing 7 M urea (17 × 21 × 0.8 mm).
The gel was run in 1× TBE buffer at 30 V/cm until the bromphenol blue
dye reached the bottom. The gel was finally fixed in 10% acetic acid,
12% methanol, dried, and exposed to x-ray film.
 |
RESULTS |
Human DNA Ligase I Inhibits DNA Elongation by pol
--
Pol
was titrated on poly(dA)200/oligo(dT)12-18,
and all reactions were carried out under conditions under which less than one nucleotide was incorporated per 3'-OH primer present, so that
statistically no more than one enzyme molecule per 3'-OH primer was
bound. Under these conditions most primers initiated no synthesis at
all, whereas DNA synthesis starting from the few primers that were
bound was the result of only one stretch of processive activity. The
length of extension of the utilized 3'-OH primers reflects the
processivity of the enzymes (50). Oligo(dT)12-18 was
hybridized to poly(dA)200 at a weight ratio of 1:10, the
equivalent of a molar ratio of 1.9:1. DNA replication reactions were
carried out with 500 ng of template DNA, corresponding to 8.9 pmol
3'-OH ends. 1, 2, 5, 10, 15, and 20 pmol of DNA ligase I, respectively, were first mixed with the required amount of PCNA (1.4 pmol) and then
added to the reaction tube. Replication was allowed to proceed for 30 min at 37 °C. Fig. 1A shows
that DNA ligase I affected DNA elongation by pol
in two different
ways. At DNA ligase I concentrations between 40 and 200 nM
the amount of nucleotides incorporated in 30 min increased by about
10-20%. An intrinsic polymerase activity of the ligase sample used
could be excluded (data not shown). At higher DNA ligase I
concentrations (0.4-0.8 µM) DNA synthesis was clearly
inhibited to an extent of 70-85%. At 50% inhibition the calculated
ratio between DNA ligase I and PCNA trimer molecules was 10:1, whereas
DNA ligase I molecules were not in excess over pol
. That the
inhibitory effect is not due to DNA ligase I binding to the 3'-OH
groups is indirectly shown in the experiment illustrated in Fig. 10
(see below). A competition experiment with an excess of primer ends
cannot be carried out because of the ability of pol
to bind to the
competitor DNA.

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Fig. 1.
A, DNA ligase I inhibits
RF-C-independent DNA replication by pol . The RF-C-independent assay
was performed as described under "Experimental Procedures," with
0.016 units of pol and 1.4 pmol of PCNA. 1, 2, 5, 10, 15, and 20 pmol of DNA ligase I, respectively, were included in the reaction from
the beginning. B, pol inhibition by DNA ligase I can be
partially alleviated by excess of PCNA. The RF-C-independent assay was
performed as described under "Experimental Procedures," with 0.03 units of pol . 1, 2, 5, 10, and 15 pmol of DNA ligase I were
included in the reaction, together with either 1.4 pmol of PCNA
(squares) or 15 pmol of PCNA (circles). 100% of
activity corresponds to the nucleotide incorporation by pol in the
absence of DNA ligase I.
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DNA Ligase I Inhibition of
Processivity Is Due to Its
Interaction with PCNA--
Levin and co-workers (41) demonstrated a
direct interaction of DNA ligase I with PCNA; furthermore, they found
that pol
,
, and
did not directly interact with DNA ligase I. We therefore next determined whether the DNA ligase I effects observed
in Fig. 1A could be attributed to an influence on pol
or
on PCNA. Fig. 1B shows that the inhibition by DNA ligase I
could be overcome with high PCNA concentrations. Addition of 15 pmol of
PCNA, which corresponds to the highest DNA ligase I amount present,
resulted in a partial reversion of the inhibitory effect. These results therefore suggest that DNA ligase I might inhibit in vitro
DNA synthesis by trapping the PCNA molecules and consequently
preventing them from interacting with pol
. pol
has very poor
processivity by itself; PCNA was established as an auxiliary factor of
pol
that dramatically increased the processivity of the latter (26, 27, 51). Gel analysis of the DNA synthesis products confirmed the loss
of processivity of pol
in the presence of DNA ligase I. Only a
decrease of long products (>200 nucleotides), but not of short
products (<200 nucleotides), was observed (data not shown).
The Inhibitory Effect of DNA Ligase I Does Not Directly Involve pol
--
From the results shown in Fig. 1B an involvement
of PCNA in the inhibitory effect by DNA ligase I can be postulated.
Therefore, we next determined whether pol
had also a role in this
process by performing an experiment in which two of the three proteins, pol
, DNA ligase I, and PCNA, were first allowed to form a possible "complex," before being exposed to the rest of the assay
components. In the experiment illustrated in Fig.
2A, PCNA was incubated with DNA ligase I for 2 min at 37 °C, and then the rest of the reaction mixture containing pol
and DNA substrate was added, and DNA synthesis was allowed to proceed for 30 min. As mentioned above, DNA
ligase I traps the PCNA molecules, thus preventing interaction with the
pol. The expected inhibitory effect (60%) was observed. In the second
case (Fig. 2B) the pol was preincubated with PCNA (2 min at
37 °C) prior to the addition of DNA ligase I and DNA. No difference
in the inhibition pattern was evident. Whether pol
and PCNA can
interact in the absence of DNA is still controversial. If this were the
case, the results would speak for a competition between pol and DNA
ligase for PCNA, whereby DNA ligase I would be the winner. On the other
hand, it would be more probable that PCNA remains free to be trapped by
DNA ligase I. In the third case (Fig. 2C), DNA ligase I and
pol
were preincubated. pol
and DNA ligase I do not interact
in vitro (data not shown and Ref. 41), and in fact DNA
synthesis was affected to exactly the same extent as above (Fig. 2,
A and B), confirming a lack of interaction
between pol
and DNA ligase I. From this experiment it can therefore
be concluded that the inhibitory effect of DNA ligase I on pol
activity might be due to DNA ligase I interaction with PCNA and does
not involve the pol itself.

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Fig. 2.
The inhibitory effect of DNA ligase I does
not directly involve pol . A, DNA ligase I in the amounts
indicated and 1.4 pmol of PCNA were first incubated together for 2 min
at 37 °C. After addition of the reaction mix containing 0.02 units
of pol and 500 ng of poly(dA)/oligo(dT), DNA synthesis was allowed
to proceed for 30 min at 37 °C and stopped as described under
"Experimental Procedures." B, 1.4 pmol of PCNA and 0.02 units of pol were incubated 2 min at 37 °C. Then DNA ligase I
(in the amounts indicated in the figure) and the reaction mix
containing the DNA were added. Reaction was carried out as above.
C, pol and various amounts of DNA ligase I were first
incubated for 2 min at 37 °C. After addition of the reaction mix
containing 1.4 pmol of PCNA, the DNA synthesis took place as described
above. D, combination of the results shown in
A-C.
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|
DNA Ligase I Does Not Affect a Sliding pol
Clamp--
In the
experiments described so far, DNA ligase I was included in the reaction
from the beginning. Therefore, the next question to answer was whether
DNA ligase I had any effect on an active, replicating pol
·PCNA
complex. DNA ligase I was added to the in vitro reaction at
different time points, and the amount of nucleotides incorporated after
2, 5, 10, 20, and 30 min was followed. Results in Fig.
3 show that inhibition of DNA synthesis
occurs if DNA ligase I is added during the first 5 min of synthesis. The inhibitory effect is not immediate, but a delay is observed, which
is directly proportional to the elapsed time between the beginning of
synthesis and addition of DNA ligase I (data not shown). When DNA
ligase I was added in the reaction after 10 or 20 min, no significant
inhibition was observed. This suggests that the DNA ligase I molecules
do not interfere with an actively moving PCNA·pol
complex, the
interaction to PCNA being limited to free trimers.

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Fig. 3.
DNA ligase I does not affect a sliding pol
clamp. 15 pmol of DNA ligase I were added to 30 µl of reaction
mix (containing 1.4 pmol of PCNA and 0.03 units of pol ) at 0, 2, 5, 10, and 20 min. 5 µl were taken out at different time points and
stopped as described under "Experimental Procedures." No addition
of DNA ligase I, squares; addition after 1 min,
diamonds; addition after 2 min, circles; addition
after 5 min, triangles; addition after 10 min,
cross; addition after 20 min, asterisks. The DNA
synthesis after 30 min in the absence of DNA ligase I was taken as
100%.
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The Inhibitory Effect of DNA Ligase I on DNA Replication by
pol
Is More Pronounced with Low Amounts of Primer
Termini--
Next, we investigated the behavior of DNA ligase I
in the presence of different ratios of primer versus
template. pol
was titrated using different template/primer ratios,
varying between a weight ratio of 25:1 and 1:1. The amount of pol
was chosen so that no more than 1 pol molecule bound per 3'-OH primer
end. Table I illustrates the different
templates used as well as the results obtained after addition of DNA
ligase I. The maximal inhibition of activity is exerted on a 25:1
template/primer base weight ratio; an increase of the number of 3'-OH
ends per poly(dA) molecule led to a decrease of the inhibitory effect.
These results might be due to the fact that more 3'-OH ends support
more active pol
·PCNA complexes, which are then inaccessible to
the DNA ligase I molecules.
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Table I
The inhibitory effect of DNA ligase I on DNA replication by pol is more pronounced at low amounts of primer termini
RF-C-independent DNA replication was carried out as described under
"Experimental Procedures" with different DNA templates. The table
illustrates the various templates used and the inhibition obtained with
20 pmol of DNA ligase I.
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DNA Ligase I Inhibits RF-C-dependent DNA Replication by
pol
on Primed M13 DNA--
RF-C-dependent DNA
elongation on singly primed M13 DNA was measured during 60 min in the
presence of DNA ligase I (Fig. 4). When
DNA ligase I was preincubated with PCNA for 2 min on ice, prior to
addition to the rest of the reaction mix, a 60% inhibition of DNA
replication was observed with 15 pmol of DNA ligase I (Fig. 4,
squares), a situation reflecting what was observed in the
RF-C independent DNA replication assay. When, on the contrary, the holoenzyme containing RF-C, PCNA, and pol
was first allowed to
assemble onto the DNA, DNA ligase I addition did not show any effect on
DNA synthesis (Fig. 4, diamonds). This suggests that the
holoenzyme, once assembled on DNA, is inaccessible to any external
action by DNA ligase I. On the other hand, when an interaction between
PCNA and DNA ligase I is allowed, DNA synthesis is inhibited as already
observed in the RF-C-independent assay (Fig. 1A).

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Fig. 4.
DNA ligase I inhibits
RF-C-dependent replication by pol . The
RF-C-dependent DNA replication on singly primed M13 DNA was
carried out as described under "Experimental Procedures." Various
amounts of DNA ligase I (1, 2, 5, 10, and 15 pmol) were either
preincubated for 2 min on ice with PCNA before addition of the missing
components (squares) or added last after holoenzyme assembly
(diamonds). DNA synthesis was allowed to proceed for 60 min
at 37 °C. Quantification of nucleotide incorporation was done as
described under "Experimental Procedures."
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DNA Ligase I Stimulates RF-C-Dependent Loading of PCNA onto Gapped
Circular DNA--
As a first step in the assembly of the holoenzyme,
RF-C loads PCNA onto DNA in an ATP-dependent manner.
Therefore, we wanted to investigate whether the presence of DNA ligase
I could affect in some way the loading of PCNA. For this, we used a
PCNA form that carried an artificial phosphorylation site for
cAMP-dependent protein kinase at its N termini (45). The
loading of the labeled PCNA onto DNA could be followed by agarose gel
electrophoresis. Fig. 5A shows
how the addition of DNA ligase I caused a strong increase in the amount
of loaded PCNA. Quantification of the autoradiogram (Fig.
5B) revealed a maximal stimulation of ~4.5 times with 2 pmol of DNA ligase I. At higher DNA ligase I concentration (10 or 15 pmol) the loading was stimulated only 2.5 times. The precise mechanism
by which the PCNA loading is stimulated remains to be investigated (see
also "Discussion"). It is surprising, however, that the reaction is
not inhibited, as would be expected in the case of an interaction of
PCNA with DNA ligase I instead of RF-C. Perhaps the reason for the
stimulation must be sought in an eventual effect of DNA ligase I on
RF-C, for example by increasing the DNA binding capacity of RF-C.

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Fig. 5.
DNA ligase I stimulates the loading of PCNA
onto DNA. A, autoradiography of the agarose gel analysis of
RF-C-dependent loading of radiolabeled PCNA onto gapped
circular DNA. The effect of various DNA ligase I amounts (1, 2, 5, 10, and 15 pmol) is shown. B, quantification of the
radioactivity co-migrating with the DNA. Loading in the absence of DNA
ligase I was designated as 1.
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DNA Ligase I Inhibits Strand Displacement Activity by pol
--
To measure the effect of DNA ligase I on strand displacement
activity by pol
(31), we used a linear, double-stranded DNA with a
defined gap of 26 nucleotides (43). Its final structure is shown in
Fig. 6A. The DNA template was
first incubated with the pol and [
-32P]dATP at
37 °C for 5 min, to allow incorporation of the first two nucleotides
(pulse). This enables the detection of a 44-nucleotide fragment. After
addition of the three missing nucleotides, synthesis was allowed to
continue for 30 min at 37 °C. Gap filling DNA synthesis results in a
70-nucleotide fragment. Strand displacement activity of pol
gives
rise to longer products of up to 344 nucleotides in length. When DNA
ligase I was added to the strand displacement reaction, an accumulation
of 70-base pair products occurred that led to a simultaneous decrease
in longer products resulting from strand displacement (Fig.
6B). These results suggested that DNA ligase I, most likely
through its interaction with PCNA, forces pol
to stop at a
5'-junction so that ligation can be performed. Such a reaction might be
important in certain DNA repair events (e.g. long-patch base
excision repair, see also "Discussion").

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Fig. 6.
DNA ligase I inhibits strand displacement
synthesis by pol . A, scheme of the 26 nucleotide gapped
dsDNA. The DNA was digested with KpnI and PvuII
before strand displacement DNA synthesis. Boldface shows the
two Ts used for pulse synthesis (see "Experimental Procedures").
B, strand displacement DNA synthesis was carried out as
described under "Experimental Procedures." Lane 1, 0.025 units of pol ; lanes 2-5, 0.025 units of pol and
increasing amounts of DNA ligase I (1, 2, 5, and 10 pmol,
respectively).
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DNA Ligase I Stimulates pol
in a PCNA-dependent
Manner--
Contrary to pol
, which in the absence of PCNA
functions very poorly incorporating only few nucleotides per binding
event (47), pol
is processive by itself and is apparently
unresponsive to PCNA on linear DNA (20, 35, 47, 52-55). However, it
was shown that PCNA increases the primer binding as well as the
nucleotide incorporation catalyzed by pol
on linear DNA, suggesting
a possible role for PCNA as a modulatory factor for pol
in various
DNA transactions (36). In the presence of RF-C and ATP, pol
can also form a stable complex with PCNA, called pol
holoenzyme (37).
On singly primed M13 DNA pol
is stimulated up to 10-fold in the
presence of PCNA, RF-C, and replication protein A (37). This template
correlated PCNA dependence suggests that pol
has a different
relationship to PCNA than pol
. As shown in Fig. 7, pol
activity was clearly affected
by the presence of DNA ligase I. 10 pmol of DNA ligase I could
stimulate nucleotide incorporation by about 100%. Product analysis on
denaturing polyacrylamide gels showed that the synthesis of all
products, independent of the length, was equally stimulated (data not
shown). Interestingly, the stimulation could be influenced by adding
PCNA. Low concentrations of PCNA (1.4 pmol were used in the experiment
shown) increased the stimulation of pol
by DNA ligase I by 20%.
High PCNA concentrations (15 pmol) did not lead to higher stimulation
but on the contrary suppressed the stimulatory effect. This could be
due to a similar effect as observed for pol
, where the DNA ligase I
inhibitory effect was overcome by an excess of PCNA. In a similar way,
PCNA could trap DNA ligase I, preventing it from acting on pol
.
Differently to what observed with pol
, in
RF-C-dependent DNA replication DNA ligase I showed no
effect on the pol
holoenzyme (Fig.
8). The kinetics of pol
stimulation
by DNA ligase I was determined on poly(dA)/oligo(dT), analogously to
pol
. DNA ligase I was added at the beginning of the reaction or
after 2, 5, 10, or 20 min. Results show that for an efficient
stimulation of pol
, addition of DNA ligase I must occur in the
first 10 min (Fig. 9). Contrary to what
was observed with pol
, the stimulatory action showed no delay, as
stimulation could be observed immediately after DNA ligase I addition
(data not shown). All together, the results obtained with pol
suggest that the action of DNA ligase I on both pols is clearly
distinguishable and implies different mechanisms.

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Fig. 7.
DNA ligase I stimulates pol in
RF-C-independent DNA replication in a PCNA-dependent
way. 2, 5, 10, and 15 pmol of DNA ligase I were added to the
replication assay at the beginning of the reaction. The reactions
included 0.02 units of pol and were carried out in the absence
(squares) or in the presence of different amounts of PCNA:
1.4 (diamonds) and 15 pmol (circles),
respectively. 100% of activity corresponded to DNA synthesis carried
out in the absence of DNA ligase I.
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Fig. 8.
DNA ligase I does not affect the pol holoenzyme. The RF-C dependent assay was performed as described
under "Experimental Procedures." Various amounts of DNA ligase I
(1, 2, 5, 10, and 15 pmol) were first preincubated with PCNA for 2 min
on ice, before addition of the missing assay components. Then the DNA
synthesis was allowed to proceed for 60 min at 37 °C.
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Fig. 9.
Kinetics of pol stimulation by DNA ligase
I. 15 pmol of DNA ligase I (dark bars) or the
corresponding volume of control buffer (light bars) were
added to the RF-C-independent assay at different time points (0, 2, 5, 10, and 20 min). The DNA synthesis was allowed to proceed for 30 min at
37 °C. Nucleotide incorporation was quantified as described under
"Experimental Procedures."
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DNA Ligase I Cannot Discriminate between pol
and pol
--
The final question we addressed was whether DNA ligase I has
any preference for pol
over pol
or vice versa. For this, DNA
synthesis was performed on poly(dA)/oligo(dT) in the presence of both
pols
and
under conditions favorable for pol
, that is at pH
6.5 and in the presence of PCNA. DNA ligase I was added at the
beginning of the reaction. Fig. 10
shows the results of the cohabitation of the four proteins, pol
,
pol
, PCNA, and DNA ligase I, in the same reaction tube.
Diamonds and circles represent the DNA synthesis
carried out by pol
and pol
, respectively, in the presence of
DNA ligase I. The lower extent of pol
inhibition is due to
different experimental conditions. DNA ligase I and DNA were in fact
preincubated prior to the addition of the other components. This
observation shows indirectly that the inhibition by DNA ligase I is not
due to its interference with the DNA substrate but that on the contrary
binding of DNA ligase I to the primer ends decreases the effect
observed. Squares show the effect of DNA ligase I when both
pols are simultaneously present in the reaction. The curve
represented with triangles derives from the addition of the
picomoles incorporated by pol
and pol
separately. Comparison of
this curve with the one where both pols are present shows that DNA
ligase I does not show any preference for one determinate pol. This
could be the case of those processes where pol
but not pol
is
required.

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Fig. 10.
DNA ligase I cannot discriminate between pol
and . RF-C-independent DNA synthesis was performed as
described under "Experimental Procedures" for pol . Various
amounts of DNA ligase I were added to a reaction mix containing 0.007 units of pol only (diamonds), 0.07 units of pol only
(circles), or 0.007 units of pol and 0.07 units of pol
together (squares). Triangles represent sum
of the values obtained with pol and pol alone (i.e.
diamonds and circles).
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DISCUSSION |
Studies on DNA ligase I suggest that it is involved in several
important cellular pathways such as DNA replication, DNA repair, and
DNA recombination (2). The intervention of DNA ligase I in these
processes implies interactions with other proteins and enzymes. A class
of enzymes that plays an essential role in various DNA transactions are
the pols. Among them, two were of particular interest for us, pol
and pol
. Both pols have been shown to play a role in DNA
replication, nucleotide excision repair, base excision repair, and
V(D)J recombination (reviewed in Ref. 17). In this study the effect of
DNA ligase I on pol
and
DNA synthesis was investigated. Our
results show that DNA ligase I can inhibit in vitro
RF-C-independent DNA replication by pol
(Fig. 1A). A
similar effect on DNA replication has been shown by Mackenney et
al. (15) where an excess of DNA ligase I inhibited in
vitro SV40 DNA replication with HeLa cell extracts. In our
experiment, carried out with purified components, the inhibition was
twice as strong, suggesting pol
and PCNA as possible targets for
DNA ligase I. Competition experiments with PCNA showed that an excess of the trimer could decrease the degree of inhibition (Fig.
1B), implying that the effect observed may be due to DNA
ligase I interaction with PCNA and not with the pol itself. Along this
line, results from Levin et al. (41) give evidence for an
interaction of the DNA ligase I with PCNA but not with pol
,
, or
. The inhibitory effect of DNA ligase I is also dependent on the
amount of 3'-ends present in the reaction. The highest level of
inhibition (71%) was obtained with a poly(dA)/oligo(dT) ratio of 25:1,
which corresponds to one primer per template molecule. An increase of
the primer/template ratio led to a decrease in the inhibitory effect
(Table I). More 3'-ends mean more active pol
/PCNA clamps,
synthesizing shorter patches of DNA. This suggested that an active
synthesizing clamp was inaccessible to the DNA ligase I. Evidence for
this hypothesis came from another experiment (Fig. 3), where the effect
of DNA ligase I on an active pol
/PCNA clamp was investigated. The
results show that inhibition of DNA synthesis was achieved only if DNA ligase I was included in the reaction within the first 5 min; once this
threshold was passed, the synthesizing complex became inaccessible for
DNA ligase I. Moreover, the inhibitory effect was not immediate after
DNA ligase I addition, again confirming the inaccessibility to an
active pol
·PCNA complex. Product analysis on denaturing
SDS-polyacrylamide gels showed a decrease in the amount of long
products, with a shift to a majority of short products. Therefore, DNA
ligase I might directly influence DNA elongation by pol
. pol
,
in contrast to pol
and
, has been shown to carry out limited
strand displacement synthesis on single-stranded DNA templates
containing two primers (31) and on gapped dsDNA (56). By using a linear
dsDNA template with a defined gap, we were able to demonstrate that DNA
ligase I inhibits the strand displacement capacity of pol
(Fig. 6).
After the gap filling reaction up to the 5'-end of the downstream DNA
fragment, pol
is usually able to displace the DNA strand, giving
rise to longer products (31). DNA ligase I inhibits the creation of
such products, enhancing the pausing of the pol at the 5'-end of the
synthesized fragment. Data with a DNA ligase I-defective human cell
line implicate this enzyme in the processing of Okazaki fragments
during DNA replication (12). In our case, DNA ligase I might recognize the single-stranded break formed by the 5'-end of the upstream product
and the 3'-end of the downstream product coming together, a situation
similar to those created by the synthesis of Okazaki fragments. Its
presence on the DNA would destabilize the pol
complex, thereby
inhibiting strand displacement activity. However, the action of DNA
ligase I on pol
does not involve any ligase activity per
se, since all the effects described so far do not require ATP
(data not shown). RF-C-dependent DNA replication is inhibited by DNA ligase I in a similar way (Fig. 4); 60% inhibition of
DNA synthesis is reached with 15 pmol of DNA ligase I. But when DNA
ligase I was added after the formation of the pol
holoenzyme, no
inhibition was observed. These results confirm what was observed before, namely that a sliding pol complex, once assembled and performing synthesis, cannot be affected by DNA ligase I. Although the
inhibitory effect observed on pol
may be due to an interaction with
PCNA, the inhibition does not affect the loading of the trimer on DNA.
On the contrary, the RF-C-dependent loading of PCNA is strongly stimulated upon addition of DNA ligase I (Fig. 5). It is very
likely that the stimulation seen is not due to the stabilization of
PCNA at the 3'-OH end by DNA ligase I, since (i) a half-life of 23 min
for PCNA on DNA has been determined (45), whereas the proteins were
fixed already after 3 min incubation (see "Experimental Procedures"); (ii) the increase in the amount of loaded PCNA is not
proportional to the increase in DNA ligase I concentration (see Fig.
5). The distinct effects of DNA ligase I on the elongation on the one
hand and on the loading on the other hand make sense, considering the
fact that PCNA has been proposed as a communicator in the different DNA
transactions (25). Stimulation of PCNA loading may be of physiological
relevance in processes such as DNA repair, where PCNA would recruit the
required proteins to the gaps or nicks. At the same time further DNA
elongation by the pol would be stopped and DNA ligase I action could
immediately take place.
The DNA ligase I effect observed on pol
was strikingly different
from what was observed with pol
. In vitro
RF-C-independent DNA synthesis was stimulated by 100% upon addition of
DNA ligase I (Fig. 7). The stimulation was influenced by PCNA in a
concentration-dependent way. High PCNA concentrations (1:1
ratio between PCNA and DNA ligase I) eliminated the stimulatory effect,
whereas low concentrations increased the stimulation. A similar effect
could also be observed in the case of pol
, where at low DNA ligase
I concentrations and in the presence of 1.4 pmol of PCNA, DNA synthesis
was stimulated by 10-20%. The reason for such an effect is unknown
but is not unique, as the human cell cycle-dependent kinase
inhibitor p21(CIP1, WAF1) shows a similar behavior at low
concentrations (57). In RF-C-dependent DNA replication, DNA
ligase I presence did not affect the pol
holoenzyme in its activity
(Fig. 8), contrary to what observed with pol
. Finally, the kinetics
of pol
stimulation on poly(dA)/oligo(dT) (Fig. 9) was different
from the one of pol
inhibition, since the stimulatory effect was
immediate.
The distinct effects of DNA ligase I on pol
and
suggest
divergent mechanisms affecting both pols. This might implicate a
different relationship between the DNA ligase I and both pol
and
pol
holoenzymes, probably due to distinct protein-protein interactions. The N terminus of DNA ligase I has no homology to other
protein sequences; therefore, it is likely to be involved in
interactions with other proteins, thereby endowing the DNA ligase I
with the specificity required in different cellular pathways. The
inhibition of pol
by DNA ligase I is probably achieved through direct interaction with PCNA. Nevertheless, an involvement of pol
in this interaction, probably through other proteins, cannot be
excluded. The PCNA function in pol
stimulation is less clear, since
the roles of pol
in vivo are not yet completely
established. As a gap filling enzyme it is apparently involved in DNA
transactions such as lagging strand synthesis, DNA repair (nucleotide
excision repair and base excision repair (38, 58)), and DNA
recombination (reviewed in Ref. 17). DNA ligase I, which is essential
for joining the Okazaki fragments, could increase the affinity of pol
for primer binding. A similar function was already suggested (36)
for PCNA; our results go along with these observations, since the
simultaneous action of DNA ligase I and PCNA led to a higher
stimulation of pol
.
We therefore speculate that in DNA replication the DNA ligase I would
control pol
DNA synthesis on the lagging strand, preventing any
strand displacement when the pol encounters the downstream DNA strand.
On the other hand, pol
, which lacks strand displacement activity,
could be recruited by DNA ligase I and PCNA to the primer ends.
Stimulation of synthesis could be of special necessity in the case of
DNA repair. In an alarm situation, the highly processive pol
would
be slowed, favoring either immediate ligation or stimulating pol
for filling the gap. In summary, our data suggest that DNA ligase I
might modulate different roles for pol
and pol
in DNA
replication or DNA repair. PCNA as an intracellular communicator (25)
could therein act as the selective partner for DNA ligase I.
We thank Robert Keller for critical reading
of the manuscript. We are grateful to Mario Amacker for providing
gapped dsDNA and for useful discussions. We thank Victoria Mackenney
and Tomas Lindahl for kindly providing DNA ligase I for the initial
experiments and Graham Daly for useful suggestions. We thank Jerard
Hurwitz for the baculovirus RF-C clones.