From the Department of Biochemistry and Biophysics,
University of Rochester School of Medicine and Dentistry, Rochester,
New York 14642 and the § Bioscience Division, Los Alamos
National Laboratory, Los Alamos, New Mexico 87545
Received for publication, February 22, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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DNA ligase I is responsible for joining Okazaki
fragments during DNA replication. An additional proposed role for DNA
ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA
ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown
here indicate that human PCNA enhances the reaction rate of human DNA
ligase I up to 5-fold. The stimulation is specific to DNA ligase I
because T4 DNA ligase is not affected. Electrophoretic mobility shift
assays indicate that PCNA improves the binding of DNA ligase I to the
ligation site. Increasing the DNA ligase I concentration leads to a
reduction in PCNA stimulation, consistent with PCNA-directed
improvement of DNA ligase I binding to its DNA substrate. Two
experiments show that PCNA is required to encircle duplex DNA to
enhance DNA ligase I activity. Biotin-streptavidin conjugations at the
ends of a linear substrate inhibit PCNA stimulation. PCNA cannot
enhance ligation on a circular substrate without the addition of
replication factor C, which is the protein responsible for loading PCNA
onto duplex DNA. These results show that PCNA is responsible for the
stable association of DNA ligase I to nicked duplex DNA.
DNA metabolism requires the coordinated activity of a multitude of
enzymes and enzyme complexes. Although the initiation of DNA
replication and DNA repair are regulated through different mechanisms,
the reactions performed to complete these pathways are similar. In
particular, Okazaki fragment processing (1) and long patch base
excision repair (2, 3) share many enzymes needed for completion of
these pathways. These include flap endonuclease 1 (FEN1),1 proliferating cell
nuclear antigen (PCNA), and DNA ligase I.
During lagging strand DNA synthesis, numerous initiator RNA primers
must be removed. The resulting gaps are filled in and sealed by
ligation to complete DNA synthesis. Two nucleases, Dna2 and FEN1, are
responsible for excising the RNA primer (4-8). Both of these enzymes
are unique structure-specific endonucleases. The preferred substrate
contains a flap structure in which the RNA primer has been displaced to
form a single-stranded tail (1, 9-13). The flap structure probably
arises as a result of displacement synthesis from an upstream Okazaki
fragment by a complex of DNA polymerase Long patch base excision repair utilizes several components common to
Okazaki fragment processing to remove bases altered by ionizing
radiation, oxidation, or alkylating agents (2, 3, 16-21). During the
repair process, an abasic site is generated after removal of a damaged
base by a DNA N-glycosylase. An apurinic/apyrimidinic endonuclease subsequently cleaves on the 5'-side of the abasic sugar to
create a nick within the DNA. Similar to the removal of initiator RNA
primers, synthesis by a DNA polymerase lifts the damaged residue and a
few additional downstream nucleotides to form a flap. As during
replication, this structure is removed endonucleolytically by FEN1
followed by ligation of the resulting nick by DNA ligase I (2, 3, 17,
19, 21). This entire process is stimulated in the presence of PCNA
(22).
PCNA is a toroidal homotrimer that is assembled around double-stranded
DNA to form a sliding clamp (23, 24). It has long been known to act as
a processivity factor for DNA polymerases by tethering the
polymerase to its template (25). However, PCNA also interacts with
other replication proteins and appears to be responsible for recruiting
these proteins to replication foci in vivo (26-28). The
interaction of PCNA and FEN1 has been examined extensively (12, 13,
29-31). The FEN1 nuclease binds to the interdomain connecting loop
region of PCNA (12, 29, 30, 32), and this association leads to a potent
stimulation of FEN1 cleavage activity (12, 13, 29). The physical
interaction between the PCNA toroid and FEN1 enhances the binding
stability of FEN1 to cleavage sites (13). In this way, PCNA serves to clamp FEN1 to its substrate in much the same way as this protein clamps
DNA polymerases to sites of DNA synthesis. The ability of PCNA to
enhance cleavage by FEN1 leads to more efficient DNA replication and
base excision repair. A physical interaction between PCNA and DNA
ligase I has also been identified (27, 30, 33).
DNA ligases have essential roles in many important cellular pathways
including DNA replication, recombination, and repair (34, 35). Of the
four DNA ligases in mammalian cells, DNA ligase I has been linked to
DNA replication (36) and base excision repair (37). This ligase has
been identified as a component of a high molecular weight replication
complex (38, 39). In addition, DNA ligase I has been shown to be
responsible for a major part of ligation activity in proliferating
cells (40-43). Cytostaining experiments with antibodies against DNA
ligase I revealed that the enzyme co-localizes in the nucleus with DNA polymerase Human DNA ligase I is comprised of a C-terminal catalytic domain and a
hydrophilic N-terminal domain. Although the N-terminal region is
dispensable for catalytic activity in vitro (50, 51), this
region is essential in vivo (52). DNA ligase I is regulated by phosphorylation at the N-terminal region of the protein (28). In
addition, the nuclear localization site of the protein has been
identified in the N-terminal domain (26, 53), and this region is also
responsible for interaction with PCNA (27).
The physical interaction between PCNA and DNA ligase I has been
characterized as a potential means by which DNA ligase I is recruited
to a replication site. Because the interaction of PCNA with FEN1
improves the catalytic rate of the nuclease, we considered here whether
the binding of PCNA to DNA ligase I also improves the efficiency of
catalysis. In this report, we initiate an investigation of the
consequences of the interaction between DNA ligase I and PCNA using
purified proteins in vitro. The advantage of this approach is that all of the observed changes in ligase function can be attributed exclusively to the presence of PCNA.
Materials--
Oligonucleotides were synthesized either by
Integrated DNA Technologies (Coralville, IA) or by Genosys
Biotechnologies (The Woodlands, TX). Radionucleotide
[ Oligonucleotide Substrates--
Oligomer sequences are listed in
Table I. The primer-template substrates
were constructed as described in the figure legends. In all substrates,
the 3'-end regions of the downstream primers share homology with the
5'-ends of their respective templates. Each upstream primer was
annealed to the proper template to create a nick between the 3'-end of
the upstream primer and the 5'-end of the downstream primer. Prior to
annealing, the 5'-radiolabeled primers were generated utilizing
[ Enzyme Assay--
The reactions containing the indicated amounts
of substrate, DNA ligase I or T4 DNA ligase, and PCNA were performed in
reaction buffer (30 mM HEPES, pH 7.6, 40 mM
KCl, 0.01% Nonidet P-40, 0.1 mg/ml bovine serum albumin, 8 mM MgCl2, and 0.1 mM ATP). The
reactions were incubated at 37 °C, terminated with 20 µl of
formamide dye (90% formamide (v/v) with bromphenol blue and xylene
cyanole), and heated to 95 °C for 5 min. After separation on a 15%
polyacrylamide, 7 M urea denaturing gel, products were
detected by PhosphorImager (Molecular Dynamics) analysis.
Phosphorylation of DNA ligase I was performed by incubating 10 fmol of
DNA ligase I with 5 × 10 Electrophoretic Mobility Shift Assay--
Reactions were
performed in binding buffer (30 mM HEPES, pH 7.6, 40 mM KCl, 0.01% Nonidet P-40, 0.1 mg/ml bovine serum
albumin, and 0.1 mM ATP) in a final reaction volume of 20 µl. After incubation at 4 °C for 15 min, products were separated
on a 1% agarose, 0.5% polyacrylamide gel in 0.25× TBE (8.9 mM Tris base, 8.9 mM boric acid, and 0.2 mM EDTA, pH 8.0) and visualized by PhosphorImager (Molecular Dynamics) analysis. The assays were performed at least in triplicate.
PCNA Stimulates DNA Ligase I Activity--
We first examined
whether the presence of PCNA influences catalysis by DNA ligase I (Fig.
1A). Lane 1 only
contains PCNA, and lane 2 only contains DNA ligase I. Titration of PCNA into the reactions (lanes 3-7) results in
a progressive stimulation of product formation. Because the only
proteins in these reactions are DNA ligase I and PCNA, the observed
enhancement of ligation activity must derive from PCNA. There is an
approximate 5-fold enhancement of ligation activity at the highest
concentration of PCNA. The addition of an unrelated protein, E. coli single-stranded DNA-binding protein, to the DNA ligase I
reaction did not result in any stimulation of ligation activity (data
not shown). All experiments were performed in excess bovine serum
albumin. The presence of this added protein did not affect DNA ligase I
activity (data not shown).
It was also important to determine the specificity of the interaction
between PCNA and DNA ligase I. Stimulation of other ligases would imply
that the mechanism is nonspecific and does not depend on contacts
between the two proteins. Therefore, PCNA was titrated into a T4 DNA
ligase reaction (Fig. 1B). The results (lanes
3-7) show no additional accumulation of ligation product. This
observation illustrates the specificity of the interaction between PCNA
and DNA ligase I.
Fig. 2A shows a time course
illustrating the activity of DNA ligase I in the absence and the
presence of PCNA. A fixed concentration of PCNA was utilized as
determined by the experiment shown in Fig. 1A. The presence
of PCNA caused a substantial stimulation of ligation activity
throughout the time course. Fig. 2B shows a second time
course demonstrating stimulation of ligation with a substrate of
different sequence and different length upstream and downstream primers
than the substrate in Fig. 2A. In both cases, the rate of
ligation was enhanced 3-4-fold.
Evidence that PCNA Enhances DNA Ligase I Binding to the Ligation
Site--
PCNA enhances the binding of various proteins to their
corresponding substrates (24). Analysis of the interaction between PCNA
and FEN1 reveals that PCNA enhances FEN1 binding stability, allowing
for greater cleavage efficiency (13). Therefore, we considered the
possibility that PCNA stimulates DNA ligase I by a similar mechanism.
We examined the effect of PCNA on DNA ligase I interaction with its
substrate using an electrophoretic mobility shift assay (Fig.
3). Incubation of the substrate with a
high concentration of DNA ligase I clearly results in the formation of
a DNA-ligase complex (lane 7). Lane 7 of Fig. 3
identifies the band corresponding to the DNA-ligase complex. Utilizing
a lower concentration of DNA ligase I, the addition of progressively higher concentrations of PCNA increased the observed amount of the
DNA-ligase complex (lanes 4-6). These results demonstrate that the binding of DNA ligase I to its substrate is enhanced by
PCNA in a concentration-dependent manner. Furthermore,
incubation of the substrate with a high level of PCNA alone failed to
result in the formation of a protein complex with DNA (lane
2). These results suggest that greater ligation efficiency is the
result of higher affinity binding of the ligase to DNA, which is
achieved through an interaction with PCNA.
Role of Phosphorylation--
DNA ligase I is a substrate for
casein kinase II (51). The N-terminal region of DNA ligase I possesses
several putative phosphorylation sites. This region contains seven
casein kinase II consensus sites, and two of these sites
(Ser66 and Ser141) have properties that are
optimal for casein kinase II phosphorylation (28). At the end of the S
phase of the cell cycle, DNA ligase I is thought to be phosphorylated
by casein kinase II (28). Although phosphorylation does not inactivate
DNA ligase I for catalysis, it prevents interaction of DNA ligase I
with the DNA replication apparatus (24). Therefore, we were interested
in determining whether phosphorylation of DNA ligase I affects PCNA stimulation of ligation activity. In Fig.
4, the addition of PCNA to reactions with
unphosphorylated DNA ligase I leads to enhanced formation of the
product (lanes 3-4). Phosphorylation of DNA ligase I
results in a slight reduction of catalytic activity (lane
6). This small reduction in activity is possibly a result of
phosphorylation itself or of the presence of casein kinase II.
Titration of PCNA into the reactions with phosphorylated DNA ligase I
does not reveal any stimulation (lanes 7 and 8).
These results demonstrate that phosphorylation of DNA ligase I prevents
PCNA from stimulating ligation activity.
Increasing DNA Ligase I Concentration Reduces PCNA
Stimulation--
To further analyze the mechanism involved in PCNA
stimulation of DNA ligase I, an enzyme titration was performed (Fig.
5). The percentage of PCNA stimulation
decreases as the concentration of DNA ligase I is increased. For
example, at 0.25 nM DNA ligase I, the addition of PCNA
leads to a 1.8 ± 0.2-fold enhancement of product formation. This
is a reduced percentage of stimulation compared with the 4.9 ± 0.4-fold enhancement at 0.05 nM DNA ligase I. Therefore,
the presence of PCNA makes the DNA ligase I molecules act as if they
were present at a higher concentration. This result indicates that PCNA
increases the rate of binding of DNA ligase I to its oligonucleotide
substrate or the rate of dissociation rather than the rate of
catalysis. In view of the results showing that PCNA enhances ligase
complex formation with DNA, the reduced stimulation is consistent with
an enhanced rate of binding.
L126D/I128E Mutant of PCNA Stimulates DNA Ligase I--
The
L126D/I128E mutant of PCNA has a severe defect in FEN1 binding ability
and a greatly reduced ability to stimulate nuclease activity (22).
Presumably, the large reduction in FEN1 binding ability prevents PCNA
from effectively stimulating catalysis by FEN1. These same mutations in
the interdomain connecting loop region of PCNA have minimal effects on
DNA ligase I binding (30). Although these mutations do not affect the
physical interaction of PCNA and DNA ligase I, they may alter PCNA
stimulation of ligase activity. In fact, the reported binding site for
DNA ligase I on PCNA is a hydrophobic pocket near the interdomain
connecting loop region of PCNA (30). To determine whether interdomain
mutations affect catalysis of ligation, we measured the ability of the
L126D/I128E mutant to increase DNA ligase I activity. In Fig.
6, DNA ligase I activity was monitored in
the absence of PCNA and in the presence of either wild-type PCNA or the
mutant PCNA. This analysis shows that both the wild-type and mutant
PCNA can stimulate DNA ligase I activity to a similar degree. This
observation supports the notion that FEN1 and DNA ligase I have partly
or completely distinct binding sites on PCNA that mediate
stimulation.
PCNA is Required to Encircle Duplex DNA to Interact Productively
with DNA Ligase I--
DNA ligase I binds to PCNA either in solution
or when the PCNA molecule is topologically linked to DNA (33).
Determining which mode of binding results in stimulation would clarify
the mechanism by which the rate of reaction is increased.
Hübscher and colleagues have shown that biotin-streptavidin
conjugations at the ends of a double-stranded linear DNA can prevent
PCNA loading (30). We employed this strategy to determine whether the
association of PCNA and DNA ligase I in solution leads to an
enhancement of ligation activity.
Fig. 7 shows the analysis of a substrate
with biotin modifications at the 5'-end of the upstream primer and the
5'-end of the template. In this way, conjugation of streptavidin to the biotinylated ends makes this substrate inaccessible to PCNA loading from the ends of the substrate. In the absence of streptavidin, the
addition of PCNA to the DNA ligase I reactions led to an enhancement of
product formation. However, the conjugation of streptavidin to the
substrate resulted in the absence of any significant stimulation upon
the addition of PCNA. This result supports the conclusion that PCNA
must encircle the substrate to effect stimulation of DNA ligase
I. When PCNA was incubated with the substrate prior to the addition of
streptavidin, there were slight enhancements in product formation at
higher concentrations of PCNA. This result is suggestive that PCNA
molecules were present on some of the substrate DNA molecules and
remained trapped there upon conjugation of streptavidin to the
substrate.
On a linear substrate, PCNA can enter the double-stranded region by
sliding over the ends (13, 29, 30). However, on a circular substrate,
PCNA requires RFC and ATP to encircle the DNA (54-58). In Fig.
8A, titration of PCNA into
reactions with a linear substrate (lanes 3-7) leads to an
enhancement of product formation. In Fig. 8B, titration of
PCNA into the reactions with a circular substrate in the absence of RFC
(lanes 3-7) does not result in any significant stimulation.
The E. coli single-stranded DNA-binding protein was also
added to minimize nonspecific interactions of either DNA ligase I or
PCNA with the single-stranded regions of DNA. Although DNA ligase I
interacts with PCNA in solution, this interaction does not lead to any
stimulation. This result shows that PCNA is required to encircle duplex
DNA to stimulate DNA ligase I activity.
RFC will open and reclose the PCNA trimeric ring around duplex DNA in
an ATP-dependent fashion (54, 56-58). The requirement of
the RFC-directed loading reaction on a circular substrate was demonstrated in Fig. 9. Lanes
1-4 are control lanes without any DNA ligase I. Lane 5 contains DNA ligase I only. The addition of PCNA (lane 6)
does not result in any enhancement of product formation. The addition
of RFC only (lane 7) also does not yield any stimulation.
However, the addition of both PCNA and RFC in conjunction with DNA
ligase I leads to the stimulation of product formation (lane
8). We interpret these results to mean that
RFC-dependent encirclement of the substrate by PCNA is
required for stimulation of DNA ligase I.
The toroidal PCNA molecule acts as a sliding clamp that
facilitates the interaction of proteins with DNA in eukaryotic systems (59). Most of the numerous proteins that bind PCNA are involved in DNA
transactions. PCNA has also been identified as the central component of
a targeting mechanism by which proteins that metabolize DNA locate
their substrates (27). DNA ligase I is required for DNA-joining
reactions that are an essential part of DNA replication and repair
pathways (37-49). Recent characterization of the binding interaction
between PCNA and DNA ligase I (30, 33) led us to investigate the effect
of this binding on the ligation reaction. We initially found that the
presence of PCNA stimulates the DNA-joining reaction catalyzed by DNA
ligase I. We further show that the stimulation reaction requires that
PCNA encircle the nicked double-stranded DNA that serves as the
ligation substrate. Additional evidence indicates that PCNA stimulates
ligation by increasing the affinity and rate of binding of DNA ligase I
to the nicked site on the substrate.
PCNA was found to stimulate DNA ligase I activity up to 5-fold when the
two purified proteins interacted in vitro. Initial reactions
were performed using a linear double-stranded DNA with a nicked site as
the substrate. It has been shown that the PCNA toroid can load onto
such substrates by diffusion over the ends (13, 29, 30). Efficient
entry requires a higher concentration of PCNA than needed for
RFC-dependent loading. An enhanced rate of ligation was
observed on substrates that differ in both sequence and length,
suggesting that the stimulation is independent of structural features
of substrates other than the requisite nick. We considered the
possibility that PCNA alters the structure of the nicked site in a way
that would facilitate the action of any DNA ligase. However, analysis
of ligation reactions with T4 DNA ligase did not reveal any stimulatory
effect. This supports the conclusion that the stimulation is related to
the specific interaction between PCNA and DNA ligase I.
The interaction between PCNA and FEN1 has been well- characterized (12,
13, 29-31). PCNA can enhance nuclease activity by increasing the
binding stability of FEN1 at its cleavage site (13). Because FEN1 and
DNA ligase I operate sequentially in some of the same pathways (1-3),
we anticipated that PCNA would stimulate DNA ligase I by a similar
mechanism. An electrophoretic mobility shift assay shows that PCNA
greatly increases the amount of DNA ligase I bound to DNA. A
supershifted complex representing PCNA and DNA ligase I bound to the
same DNA substrate is conspicuously absent. After facilitating the
binding of DNA ligase I, PCNA must dissociate either before or during
the electrophoresis step of the mobility shift assay. The DNA ligase I
dissociation rate could be substantially lower than that of PCNA so
that the ligase is retained during the movement of the complex on the
gel. It is noteworthy that electrophoretic mobility shift assays
showing enhancement of FEN1 binding by PCNA also have no
supershifted complex (13).
Another interesting possibility is that PCNA induces a conformational
change in DNA ligase I that enhances the binding stability of this
enzyme to the ligation site. A recent study has shown that FEN1
undergoes a conformational change upon binding to a flap DNA substrate
(60). If binding to PCNA can induce this conformational change, then
the addition of PCNA may lead to the stable association of a larger
population of FEN1 nucleases to the substrate. This would allow FEN1 to
be retained on the substrate during electrophoresis even after PCNA
dissociates. If DNA ligase I operates in a similar manner, the addition
of PCNA would lead to a greater population of DNA ligase I molecules
that are bound to the nicked duplex DNA. The stable association
resulting from a conformational change would allow retention of DNA
ligase I on the DNA in the absence of PCNA during electrophoresis.
Another important observation is that adding PCNA has the same effect
on DNA ligase I binding and catalysis as increasing the concentration
of ligase. In addition, at high concentrations of DNA ligase I, PCNA
can no longer substantially improve the rate of the reaction. These
results support the conclusion that the observed stimulation is the
result of an increased rate of binding of DNA ligase I to the substrate
DNA. Because the binding rate is maximized at high ligase
concentrations, no further rate enhancement is possible.
Considering that the stimulatory effect appears to be a result of the
physical interaction between PCNA and DNA ligase I, inhibiting this
interaction should prevent stimulation. This was clearly illustrated
when DNA ligase I was phosphorylated by casein kinase II. During the
cell cycle, casein kinase II has been proposed to function in
phosphorylating DNA ligase I to abolish the interaction of this protein
with the replication machinery (24). Casein kinase II phosphorylates
several residues within the N-terminal region of DNA ligase I (28).
This region is not involved in catalysis but is required for binding to
PCNA (27). Our experiments show that incubation of DNA ligase I with
casein kinase II has little effect on the activity of the purified DNA
ligase I. However, phosphorylation of DNA ligase I completely
eliminates the stimulatory effect by PCNA.
Previous studies have shown that DNA ligase I can bind to PCNA in
solution or PCNA that is topologically linked to DNA (33). However,
functional interactions of other proteins with PCNA have always
involved PCNA that is encircling duplex DNA (13, 23-25, 30, 61). Our
results show no exception to this rule. Blocking the entrance of PCNA
onto a linear substrate with biotin-streptavidin moieties effectively
prevented stimulation. In addition, PCNA could not stimulate DNA ligase
I activity on a circular substrate unless the PCNA molecules were
loaded onto the substrate by the ATP-dependent action of
RFC. Therefore, these observations support the requirement for PCNA to
surround the duplex DNA to augment DNA ligase I activity.
Although both FEN1 and DNA ligase I are stimulated by PCNA, these two
enzymes have different binding sites on PCNA (30). FEN1 association
occurs through the interdomain connecting loop region of PCNA (12, 29,
30, 32), whereas DNA ligase I interacts with a hydrophobic pocket near
the interdomain connecting loop (30). Our analysis of a PCNA mutant
with a severe defect in FEN1 binding activity and a nuclease
stimulation deficiency shows that this mutant is still capable of
interacting with DNA ligase I to effectively stimulate ligation
activity. Another group has shown that mutations in the interdomain
connecting loop region of PCNA do not have significant effects on DNA
ligase I binding (30). Evidently, these mutations also do not interfere
with the stimulation of DNA ligase I activity. The results with this PCNA mutant further support the differentiation of PCNA interactions with FEN1 and DNA ligase I. FEN1 and DNA ligase I operate sequentially in DNA replication and repair pathways (1-3). We are currently considering whether the different binding sites are utilized for a
switching process by which one of the enzymes associates with the PCNA
molecule while the other enzyme dissociates.
Other studies have supported a potential switching mechanism whereby
different proteins interact with PCNA (14, 23, 24, 27, 59, 62). DNA
ligase I inhibits DNA synthesis in vitro by polymerase All of these results enlighten our understanding of the final steps of
Okazaki fragment processing and the DNA excision repair pathways. PCNA
functions to recruit the relevant enzymes to their sites of
interaction. A revised model of Okazaki fragment processing (Fig.
10) shows that PCNA is first brought to
a FEN1 cleavage site by the action of a polymerase. Strand displacement
synthesis leads to the generation of a flap substrate for FEN1.
Dissociation of the polymerase from PCNA allows FEN1 binding to occur.
After FEN1 cleaves the flap structure, subsequent nuclease dissociation
permits DNA ligase I binding to PCNA. In this way, all of the pertinent proteins are brought to a replication fork or a repair site through mediated interactions with PCNA. The exact mechanisms whereby the
various associations and dissociations are orchestrated are not yet
known. Additional studies have to be performed to further clarify these
sequential interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and its accessory factors,
PCNA and replication factor C (RFC) (14). Dna2 is thought to cleave
beyond the RNA segment within the tail, and the remaining displaced DNA
is removed by FEN1 (5-7). Finally, the two fragments are joined
through ligation by DNA ligase I (1, 15).
, implicating DNA ligase I in DNA replication (44). A DNA
ligase I mutant human cell line, 46BR, exhibits abnormal joining of
Okazaki fragments (45-48), but the replication defect in extracts from
this cell line can be complemented by the addition of DNA ligase I
(49). A recent study with the DNA ligase I mutant cell line 46BR.1G1
reveals that the interaction between PCNA and DNA ligase I is integral
to coordinating the ligation steps that complete long patch base
excision repair (37). These observations imply an important role for
DNA ligase I in DNA replication and repair.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer
Life Sciences. The T4 polynucleotide kinase and T4 DNA ligase were from
Roche Diagnostics. Casein kinase II was obtained from Roche Molecular Biochemicals. Escherichia coli single-stranded DNA-binding
protein was obtained from Promega. Micro Bio-Spin 30 chromatography
columns were from Bio-Rad. All other reagents were the best available commercial grade. Recombinant human DNA ligase I (19) and recombinant human PCNA (13) were prepared as described previously. Purified DNA
ligase I was dialyzed into a storage buffer (30 mM HEPES, pH 7.6, 10% glycerol, 15% sucrose, 25 mM KCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, and 1 mM EDTA) and stored at
80 °C. Purified PCNA was
dialyzed into a storage buffer (30 mM HEPES, pH 7.6, 20%
glycerol, 30 mM KCl, 1 mM dithiothreitol,
0.01% Nonidet P-40, and 1 mM EDTA) and stored at
80 °C.
-32P]ATP and T4 polynucleotide kinase according to
the manufacturer's instructions. Unincorporated radionucleotides were
removed with Micro Bio-Spin 30 chromatography columns. All radiolabeled
primers were purified by gel isolation from a 15% polyacrylamide, 7 M urea denaturing gel prior to annealing. Substrates were
annealed by mixing 2 pmol of the respective downstream primer with 5 pmol of the corresponding template in annealing buffer (10 mM Tris base, 50 mM KCl, and 1 mM
EDTA, pH 8.0) to a final volume of 30 µl. The mixtures were heated to
95 °C for 5 min and allowed to cool to room temperature. A
corresponding upstream primer (10 pmol) was subsequently added and
annealed by incubating at 37 °C for 1 h. The circular substrate
was generated by annealing the downstream primer (D2), the
template (pBS(+)), and the upstream primer (U3) at a molar
ratio of 1:2.5:5, respectively. The mixture was heated to 95 °C for
5 min and subsequently cooled to room temperature.
Oligonucleotide sequences (5'-3')
3 milliunits of casein
kinase II at 30 °C for 10 min (0.1 mM ATP). For the
biotin-streptavidin assay, the substrate was incubated with PCNA either
before or after the addition of streptavidin. Conjugation of
streptavidin (added in a 50-fold molar excess over substrate) to the
biotinylated substrate was accomplished by placing the reactions at
4 °C for 10 min. These reactions contained 1 fmol of DNA ligase I. The reactions utilizing human RFC were performed in a buffer containing
30 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.01% Nonidet P-40, 0.1 mg/ml bovine serum albumin, 1 mM
dithiothreitol, 8 mM MgCl2, and 0.1 mM ATP. All assays were performed at least in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PCNA stimulates DNA ligase I activity, but
PCNA does not stimulate T4 DNA ligase. Reactions of 20 µl
containing 20 fmol of DNA substrate were performed as described under
"Experimental Procedures." The following amounts of PCNA were added
(as indicated by the triangles): 0.5, 1.0, 1.5, 2.0, and 2.5 pmol. The reactions were incubated at 37 °C for 15 min. Substrate
and ligation product sizes are as indicated. The substrate is comprised
of D3:U4:T4 with a
-32P radiolabel at the 5'-end of the downstream primer.
The conversion of substrate to product was determined by quantitating
the substrate and product utilizing PhosphorImager (Molecular Dynamics)
analysis. A, analysis of ligation activity using 5 fmol of
DNA ligase I per reaction (lanes 2-7). Upon the addition of
PCNA, the approximate stimulation levels are 1.7-, 2.5-, 3.6-, 4.4-, and 5.0-fold, respectively. B, analysis of ligation activity
using 2 × 10
3 milliunits of T4 DNA ligase per
reaction (lanes 2-7). LIG. I, DNA ligase I;
T4 LIG., T4 DNA ligase; nt, nucleotide.
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Fig. 2.
Enhancement of DNA ligase I activity by PCNA
is consistent over time and with different substrates. Reactions
of 140 µl containing 140 fmol of DNA substrate and 35 fmol of DNA
ligase I were performed as described under "Experimental
Procedures." The reactions with PCNA contained 17.5 pmol. The
reactions were incubated at 37 °C, and 20-µl aliquots were removed
at 0, 1, 3, 5, 7, 10, and 15 min as indicated by the
triangles. Substrate and ligation product sizes are as
indicated. The 5'-end of each downstream primer was radiolabeled with
-32P. The conversion of substrate to product was
determined by quantitating the substrate and product utilizing
PhosphorImager (Molecular Dynamics) analysis. A, product
analysis of a substrate comprised of
D3:U4:T4. The approximate
stimulation levels upon PCNA addition from 1 to 15 min are 2.6-, 3.5-, 3.9-, 3.9-, 4.1-, and 4.0-fold, respectively. B, product
analysis of a substrate containing
D1:U1:T1. The approximate
stimulation levels upon PCNA addition from 1 to 15 min are 2.8-, 3.5-, 3.6-, 3.5-, 3.2-, and 3.1-fold, respectively. nt,
nucleotide.
View larger version (26K):
[in a new window]
Fig. 3.
The addition of PCNA increases the formation
of the DNA-ligase complex. Reactions of 20 µl containing 5 fmol
of DNA substrate and 5 fmol of DNA ligase I were performed as described
under "Experimental Procedures" (lanes 3-6). The
reactions were incubated at 4 °C for 15 min. PCNA concentrations of
0.625, 1.25, and 2.50 pmol were utilized as denoted by the
triangle. Lanes 1, 2, 3, and
7 are control lanes. The reaction in lane 1 only
contains substrate. The reaction in lane 2 was performed in
the absence of DNA ligase I and in the presence of PCNA. The reaction
in lane 3 contains DNA ligase I without PCNA. Lane
7 represents a reaction with 5 fmol of DNA substrate and 20 fmol
of DNA ligase I. The substrate is comprised of
D2:U3:T3. LIG. I, DNA
ligase I.
View larger version (51K):
[in a new window]
Fig. 4.
Phosphorylation of DNA ligase I by casein
kinase II eliminates PCNA stimulation. Reactions of 20 µl
containing 5 fmol of DNA substrate and 1 fmol of DNA ligase I were
performed as described under "Experimental Procedures" (lanes
2-4 and 6-8). PCNA concentrations of 0.25 and 0.50 pmol were utilized as denoted by the triangles. The
substrate is comprised of D2:U3:T3.
The reactions were incubated at 37 °C for 10 min. Substrate and
ligation product sizes are as indicated. The 5'-end of the downstream
primer was radiolabeled with -32P. Lanes 2-4
represent an analysis of ligation activity with untreated DNA ligase I. Lanes 6-8 depict an analysis of ligation activity with DNA
ligase I that has been treated with casein kinase II. Phosphorylation
of DNA ligase I by casein kinase II was performed as described under
"Experimental Procedures." CKII, casein kinase II;
LIG. I, DNA ligase I; nt, nucleotide.
View larger version (43K):
[in a new window]
Fig. 5.
The level of PCNA stimulation decreases at
high concentrations of DNA ligase I. The reactions were incubated
at 37 °C for 5 min. Substrate and ligation product sizes are as
indicated. The 5'-end of the downstream primer was radiolabeled with
-32P. Reactions of 20 µl containing 5 fmol of DNA
substrate and 1, 3, or 5 fmol of DNA ligase I were performed as
described under "Experimental Procedures." The reactions in the
presence of PCNA contained 0.5 pmol of PCNA. The addition of PCNA leads
to stimulation levels of 4.9 ± 0.4-fold (0.05 nM DNA
ligase I), 2.3 ± 0.2-fold (0.15 nM DNA ligase I), and
1.8 ± 0.2-fold (0.25 nM DNA ligase I). The narrow
standard deviations support the statistical relevance of the
suppression of stimulation at high ligase concentrations. The substrate
is comprised of D2:U3:T3.
LIG. I, DNA ligase I; nt, nucleotide.
View larger version (40K):
[in a new window]
Fig. 6.
The L126D/I128E mutant of PCNA enhances DNA
ligase I activity. Reactions of 140 µl containing 140 fmol of
DNA substrate and 35 fmol of DNA ligase I were performed as described
under "Experimental Procedures." In the reactions with PCNA, 17.5 pmol of either wild-type or mutant PCNA was utilized. The reactions
were incubated at 37 °C, and 20-µl aliquots were removed at 0, 1, 3, 5, 7, 10, and 15 min as indicated by the triangles.
Substrate and ligation product sizes are as indicated. The 5'-end of
the downstream primer was radiolabeled with -32P. The
substrate is comprised of D3:U4:T4.
nt, nucleotide.
View larger version (23K):
[in a new window]
Fig. 7.
The addition of streptavidin blocks PCNA
loading onto the substrate, inhibiting stimulation of ligation.
The substrate is comprised of
D1:U2:T2. The 5'-end of the
upstream primer and the 5'-end of the template were biotinylated.
The 5'-end of the downstream primer was radiolabeled with
-32P. Reactions of 20 µl containing 5 fmol of DNA
substrate and 1 fmol of DNA ligase I were performed as described under
"Experimental Procedures." PCNA concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 pmol were utilized. Streptavidin was conjugated to the
biotinylated 5'-ends according to "Experimental Procedures." The
reactions were incubated at 37 °C for 10 min. The conversion of
substrate to product (%) was determined by quantitating the substrate
and product on a denaturing polyacrylamide gel by PhosphorImager
(Molecular Dynamics) analysis.
View larger version (32K):
[in a new window]
Fig. 8.
PCNA does not stimulate DNA ligase I activity
on a circular substrate. The reactions were incubated at 37 °C
for 10 min. Substrate and ligation product sizes are as indicated. The
5'-end of the downstream primers were radiolabeled with
-32P (as indicated by the asterisks). Reactions of 20 µl containing 5 fmol of DNA substrate were performed as described
under "Experimental Procedures." PCNA concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4 pmol were utilized as denoted by the triangles.
A, analysis of a linear substrate
(D2:U3:T3). The reactions contained
0.8 fmol of DNA ligase I. B, analysis of a circular
substrate (D2:U3:pBS(+)). E. coli
single-stranded DNA-binding protein (0.25 pmol) was added to coat the
single-stranded regions of the substrate. The reactions contained 0.2 fmol of DNA ligase I. LIG. I, DNA ligase I; nt,
nucleotide.
View larger version (52K):
[in a new window]
Fig. 9.
RFC is required to load PCNA onto a circular
substrate to affect DNA ligase I activity. The substrate is
comprised of D2:U3:pBS(+). The reactions were
incubated at 37 °C for 10 min. Substrate and ligation product sizes
are as indicated. The 5'-end of the downstream primer was radiolabeled
with -32P. Reactions of 20 µl containing 5 fmol of DNA
substrate and 0.2 fmol of DNA ligase I were performed as described
under "Experimental Procedures" (lanes 5-8). E. coli single-stranded DNA-binding protein (0.25 pmol) was added to
coat the single-stranded regions of the substrate. The reactions in the
presence of PCNA contained 50 fmol of PCNA, and the reactions in the
presence of RFC contained 15 fmol of RFC. Lanes 1-4
represent control lanes without DNA ligase I. LIG. I, DNA
ligase I; nt, nucleotide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(14). However, this inhibition is overcome by the addition of a high
concentration of PCNA (14). This observation suggests that inhibition
of DNA synthesis by DNA ligase I is a result of a binding competition
between the polymerase and ligase for PCNA. Analysis of an active
polymerase
-PCNA complex reveals that the inhibitory effect of DNA
ligase I is not as evident, suggesting that an active polymerizing
complex may be inaccessible to DNA ligase I (14). These observations
hint at a switching mechanism whereby inactivation of the polymerase
complex, possibly by the encounter with a downstream primer, frees PCNA
for interaction with other proteins such as FEN1 and DNA ligase I. In
this way, PCNA may act as a sequential target for specific proteins at
their respective substrates.
View larger version (47K):
[in a new window]
Fig. 10.
A model of Okazaki fragment processing.
RFC is responsible for loading PCNA onto the duplex DNA in an
ATP-dependent manner. PCNA initially serves as a sliding
clamp for DNA polymerase . This polymerase complex displaces a
portion of the downstream Okazaki fragment. RPA binds to the
single-stranded regions. Dna2 and FEN1 function to remove the initiator
RNA and the displaced DNA. FEN1 binds to PCNA to perform cleavage.
Subsequently, DNA ligase I binds to PCNA to seal the nick.
This is the first report of PCNA stimulation of DNA ligase I activity. Although several groups have characterized the binding interaction between PCNA and DNA ligase I (27, 30, 33, 37), no stimulatory effect has previously been observed to result from this interaction. Observations have included the absence of any effect (33) and an inhibition of ligation (30). Differences in experimental conditions, including the concentrations of proteins and the types of buffers employed, may explain the discrepancies that have been reported.
In summary, we present evidence that human PCNA stimulates the activity
of DNA ligase I, and the stimulatory effect is a result of PCNA
stabilization of DNA ligase I binding to the ligation site on its
oligonucleotide substrate. Stimulation occurs on a variety of ligation
substrates, providing that these substrates have topologically-linked
PCNA. Accordingly, the mechanism of stimulation can be explained by a
binding interaction between a PCNA molecule encircling the duplex DNA
and a DNA ligase I molecule at the ligation site as depicted in our
proposed model.
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ACKNOWLEDGEMENTS |
---|
We are grateful to the members of the Bambara laboratory for insightful discussions. We thank Dr. Hirobumi Teraoka for providing a human DNA ligase I expression plasmid (phLigI) and Dr. Michael S. DeMott for the purification of recombinant human DNA ligase I. In addition, we thank Dr. Vladimir N. Podust for kindly providing purified human replication factor C.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM24441 and in part by Grant GM59301 (to M. S. P.) from the National Institutes of Health.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: Univ. of Rochester Medical Center, Dept. of Biochemistry and Biophysics, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 716-275-3269; Fax: 716-271-2683; E-mail: robert_bambara@urmc.rochester.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M101673200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FEN1, flap endonuclease 1; PCNA, proliferating cell nuclear antigen; RFC, replication factor C.
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