(Received for publication, February 2, 1995; and in revised form, May 25, 1995)
From the
Proliferating cell nuclear antigen (PCNA) is essential for
eukaryotic DNA replication and functions as a processivity factor of
DNA polymerase (pol
). Due to the functional and structural
similarity with the
-subunit of Escherichia coli DNA
polymerase III, it has been proposed that PCNA would act as a molecular
clamp during DNA synthesis. By site-directed mutagenesis and
biochemical analyses, we have studied the functional domains of human
PCNA required for stimulation of replication factor C (RF-C) ATPase and
DNA synthesis by pol
. Short deletions from either the N or C
termini caused drastic changes in extraction and chromatographic
behaviors, suggesting that both of these terminal regions are crucial
to fold the tertiary structure of PCNA. The short C-terminal stretch
from Lys
to Glu
is necessary for
stimulation of RF-C ATPase activity, but not for stimulation of DNA
synthesis by pol
. Nine basic amino acids that are essential for
activating DNA synthesis by pol
are positioned at the internal
-helices of PCNA. This result is in good agreement with the
observation that PCNA has a ring structure similar to the
-subunit
and clamps a template DNA through this positively charged internal
surface. Several other charged amino acids are also required to
stimulate either RF-C ATPase or pol
DNA synthesis. Some of them
are positioned at loops which are exposed on one of the side surface of
PCNA adjacent to the C-terminal loop. In addition, the
-sheets
composing the intermolecular interface of the trimeric PCNA are
important for interaction with pol
. Therefore, the outer surface
of PCNA has multiple functional surfaces which are responsible for the
interaction with multiple factors. Furthermore, the two side surfaces
seem to be functionally distinguishable, and this may determine the
orientation of tracking PCNA along the DNA.
The proliferating cell nuclear antigen (PCNA) ()is an
essential replication factor for simian virus 40 (SV40) DNA replication in vitro and is involved in the elongation stages of DNA
replication(1, 2, 3, 4) . Recent
studies have further demonstrated the involvement of PCNA in cellular
chromosomal DNA replication in
vivo(5, 6, 7) . In addition to its
essential role in DNA replication, PCNA is required for nucleotide
excision repair of DNA (8, 9, 10) and also
may participate in the cell cycle control as demonstrated by an
interaction with a cyclin dependent kinase complex(11) .
Therefore, PCNA is multifunctional through the interaction with several
specific partners, and all of the functions are crucial for cell
proliferation.
Genes encoding PCNA have been isolated from various
eukaryotes and are composed of highly conserved amino acid sequences of
around 260 residues. The functions of PCNA during DNA replication have
been elucidated by studies of SV40 DNA replication in vitro.
In this reaction, three protein components, PCNA, DNA polymerase (pol)
, and replication factor C (RF-C) are required for leading strand
DNA synthesis following the initiation of DNA synthesis by DNA pol
at the SV40 origin(12) . PCNA and RF-C form a complex at
a primer-template junction(13) , and consequently, the DNA
synthesizing DNA polymerase switches from pol
to pol
(14) . In this process, PCNA makes connections between the
primer-end recognition reaction and the DNA synthesis reaction through
physical interactions with RF-C and pol
. These interactions have
been demonstrated as the stimulation of RF-C ATPase and pol
DNA
synthesis by PCNA(15) . However, little is known about the
detailed mechanism of how such a complex is formed and which structures
are responsible for these interactions.
The assembly of a functional
replication DNA polymerase holoenzyme through protein-protein
interaction has been reported for Escherichia coli DNA pol III
and bacteriophage T4 DNA polymerase (for a review, see (16) and (17) ). The first step is a binding of an
ATPase complex (E. coli complex and bacteriophage T4
gene 44/62 protein complex) to a primer-template junction. Following
this process, a processivity factor (E. coli
-subunit and
bacteriophage T4 gene 45 protein) forms a secondary complex with the
ATPase. This complex tethers its specific core DNA polymerase (E.
coli
,
,
, and bacteriophage T4 gene 43 protein)
onto a DNA template mediated by hydrolysis of ATP, and a processive DNA
polymerase holoenzyme is formed. Judging from the highly conserved
reaction processes, there should be close structural and functional
relationships between these bacterial and eukaryotic replication
factors. The three-dimensional structure of pol III
-subunit has
been determined by x-ray crystallography(18) . The tertiary
structure of
-subunit is a characteristic ring or torus-like
structure composed of two homologous subunits that combine to form a
DNA tracking protein (for a review, see (19, 20, 21) ). The central hole in the
structure has a highly positively charged surface containing six
-helices which are thought to be important for interaction with
the DNA template. The outer surface has several
-sheet structures
that may interact with other protein components. This model proposes
three structurally related units in each monomer subunit and therefore,
the whole
-subunit dimer consists of six of these units.
Structural similarity between PCNA and the
-subunit has been
suggested, even though their primary sequences are not
related(18) . The prediction has been realized to be
essentially correct by a recent study of the crystal structure of PCNA
from Saccharomyces cerevisiae(22) . Based on the
crystallography results, each PCNA monomer consists of two structurally
related domains and the whole molecule contains three PCNA subunits.
Thus, like the
-subunit of pol III, the PCNA clamp has six
structurally related units.
To study the assembly of these important
eukaryotic replication factors into a functional complex, detailed
information on the molecular structure and function of PCNA and the
mechanism of protein-protein interactions with RF-C and pol is
necessary. We therefore constructed mutations covering the whole PCNA
gene by site-directed mutagenesis. Each mutant PCNA protein was
expressed in E. coli cells and purified. Stimulation of RF-C
ATPase and pol
activity was then compared to the wild type
protein.
Figure 1:
Mutation sites in aligned amino acid
sequences of human(32) , rice(33) , Drosophila
melanogaster (34), yeast (S. cerevisiae) (6) PCNA. Conserved amino acids, which were substituted to
alanine in this study, were indicated by filled boxes for
basic amino acids and shaded boxes for acidic amino acids.
Regions corresponding to -sheets and
-helices (
A1 to
I2, and
A1 to
B2,
respectively) follow the labeling of the crystal structure of yeast
PCNA(22) .
The mutated DNA sequences were confirmed with an Applied Biosystems model 373A automatic DNA sequencer (Applied Biosystems) or a conventional electrophoresis method with T7 DNA polymerase Version 2.0 Kit (U. S. Biochemical Corp., Cleveland, OH).
For mutant PCNAs, K20A,
D189A, E192A, R210A, and D257A, the pooled Econo-pac Q cartridge
fractions was loaded onto a 5-ml Econo-pac HTP cartridge (Bio-Rad)
equilibrated with buffer H (20 mM KPO (pH 6.9),
0.01% Nonidet P-40, 10% glycerol, 2 µg/ml leupeptin, 1 mM
PMSF, and 1 mM DTT). Proteins were eluted with a 30-ml
gradient of 0.02 to 0.6 M KPO
. PCNA-containing
fractions were pooled, mixed with an equal volume of 4 M (NH
)
SO
, and loaded onto a 5-ml
Econo-pac methyl HIC cartridge (Bio-Rad) equilibrated with buffer A
containing 2 M (NH
)
SO
.
Proteins were eluted with a 30-ml linear gradient from 2 to 0 M (NH
)
SO
in buffer A.
PCNA-containing fractions around 0.3 M
(NH
)
SO
were pooled and dialyzed
against buffer A with 50 or 100 mM NaCl and loaded onto a Mono
Q (HR 5/5) as above.
In order to investigate functional regions of
PCNA, 33 mutations were introduced into the PCNA gene in the plasmid
pT7PCNA, and the mutant PCNA proteins were expressed as for the wild
type. Several N- or C-terminal truncations and amino acid substitution
mutations at highly conserved amino acids were designed to study.
Terminal deletions were designed to determine which region of the PCNA
was functionally important. An N-terminal deletion mutant,
2-9 that lacks 8 amino acids from the second codon, and
three C-terminal deletion mutants,
257-261,
254-261, and
250-261 lack 5, 8, and 12 amino
acids from the C terminus, respectively, were constructed. The amino
acids in PCNA highly conserved among several species were expected to
be involved in its indispensable functions. Furthermore, since a
substitution of a charged amino acid to a noncharged one will have a
minimal effect on the whole structure of PCNA, any altered phenotype
may simply represent an altered function rather than the denaturation
of the whole protein. Twenty-nine charged amino acids as indicated in Fig. 1were individually substituted with the noncharged amino
acid, alanine.
In all cases, the mutant PCNA proteins accumulated to
high amounts in the E. coli cells after the induction of the
T7 promoter and became major components as observed for the wild type
PCNA. Most of the mutant PCNAs, except for 2-9 and
250-261, were extracted efficiently from the E. coli cells by a sonic extraction and behaved as a single component of
native molecular mass of around 120 kDa, as determined by size
fractionation chromatography during the purification. This molecular
mass is in good agreement with the prediction that PCNA is in a
trimeric state(22) .
Figure 2:
RF-C ATPase assay with mutant PCNAs. Time
course of P production with 4 ng of RF-C and 280 ng of
mutant PCNAs is shown. Mutant PCNAs are shown with symbols in the inset. Each graph (A-E) demonstrates a result
of an independent experiment with controls with and without wild type
PCNA (filled and open circles,
respectively).
Figure 3:
Pol activity with mutant PCNAs. DNA
synthesis was measured with 0.2 unit of pol
and increasing
amounts of wild or mutant PCNAs as described under ``Materials and
Methods.'' The incorporated [
-
P]TMP
after an incubation at 37 °C for 15 min is shown. Mutant PCNAs
added are indicated with symbols in each inset. Each graph
shows a result of an independent experiment and has an activity with
wild type PCNA (filled circles). Picomoles of the incorporated
dTMP in 1 µl of a reaction mixture are
shown.
The activity
pol DNA synthesis was assayed with the DNA polymerase, PCNA, and
a template DNA, poly(dA)
oligo(dT). Under our assay condition, due
to the presence of excess primer ends on poly(dA)
oligo(dT), pol
is able to carry out processive DNA synthesis without RF-C, and
the pol
activity may be dependent on one or two of the following
functions of PCNA, direct protein-protein interaction between PCNA and
pol
and the DNA clamping activity by PCNA. The mutant data do not
discriminate between these two functions.
In the case of deletion
mutants 2-9 and
250-261, which have lost the
native configuration, they did not exhibit the stimulative activities
for RF-C ATPase and pol
DNA synthesis (Fig. 2A and Fig. 3A for
250-261, but data is
not shown for
2-9). The loss of the stimulative activity was
not simply due to the denaturation of this protein with urea during
extraction, but because of an inability to fold into the native
structure by these truncations, inasmuch as the wild type PCNA could be
renatured and become fully active by removing urea after being
extracted by the same procedure as these mutants (data not shown).
Therefore, the native trimeric structure is necessary for both the
functions.
Among the conserved amino
acids in the PCNA gene, 14 basic residues were substituted with Ala
individually. Mutants K13A, K14A, K20A, K77A, R146A, R149A, and K217A
lost the stimulation of pol activity and exhibited only a basal
level DNA synthesis. The addition of K80A, K110A, or R210A to the pol
DNA synthesis reaction caused a limited stimulation in pol
activity at less than 50% of wild type (Fig. 3D). With
mutants R64A and K168A, a partial decrease in stimulation,
corresponding to 50-80% of wild type activity, was observed (Fig. 3, C and E). The rest of the basic amino
acid substitutions did not affect the stimulatory activity for pol
DNA synthesis, showing that these residues were not required for
the activity (data not shown except for K254A in Fig. 3).
One
characteristic feature of both the pol III -subunit and yeast PCNA
is the presence of
-helices with positively charged amino acids at
the inner surface of the ring(18, 22) . This
structural feature may be responsible for the DNA clamping activity of
these DNA tracking proteins. One subunit of PCNA has 4
-helices,
and each has 2 or 3 basic residues which are highly conserved from
human to yeast (Fig. 1). Lys
, Lys
,
Lys
, Lys
, Lys
,
Arg
, Arg
, and Lys
are exactly
positioned at each
-helix and all are necessary to stimulate pol
activity. These contribute to the positive electrostatic
potential in the center of the ring and are suggested to be necessary
for an interaction with DNA. Thus, their requirement for pol
activity may be a reflection of such an interaction with the template
DNA. It is of interest that the elimination of only 1 residue out of 9
positively charged residues reduces drastically the ability of PCNA to
stimulate pol
activity. Probably, a certain positive
electrostatic distribution on the inner surface is important to
maintain the DNA clamping activity.
The inability to stimulate pol
DNA synthesis with these mutant PCNAs may due to the decrease in
either the initial DNA clamping activity or the sliding activity on
DNA. To distinguish the mechanisms, we analyzed the products from DNA
synthesis by pol
using denatured alkaline agarose gel
electrophoresis (Fig. 4). A full-length product of 200-300
nucleotides long was synthesized with wild type PCNA but DNA shorter
than 20 nucleotides was produced without PCNA. If we analyzed the
reaction products with mutants K13A, K14A, K20A, K77A, and K217A, all
of them were significantly processive, although their incorporation of
dTMP was only a few percent of the fully stimulated pol
DNA
synthesis ( Fig. 3and Fig. 4). Since the incorporation is
corresponding to a DNA synthesis of less than 0.3 nucleotide from each
primer end, length of the product represents the processivity of DNA
synthesis. Therefore, these mutants block mainly the initiation of pol
DNA synthesis but not the processivity of the DNA synthesis. This
implies that the elimination of positive charges in the
-helices
may decrease the DNA clamping activity of PCNA but not the sliding
activity, leading to inefficient initiation of pol
DNA synthesis.
Figure 4:
Product analysis of pol DNA
synthesis with mutant PCNAs by alkaline agarose gel electrophoresis.
DNA synthesis reaction was carried out as described under
``Materials and Methods'' with indicated mutant PCNA
proteins. In reactions from minus to K217A and from K80A to R210A,
TMP of 10 times and five times higher
specific activities than that of the rest were used, respectively. The
products of roughly same radioactivity were loaded onto a 1% agarose
gel and separated in an alkaline condition as described in Tsurimoto
and Stillman(14) . Length of DNA (bases) obtained from a size
marker is shown at the left side.
None of the basic amino acids in the internal -helices affected
stimulation of RF-C ATPase ( Fig. 2and data not shown). Taking
the complex formation among RF-C, PCNA, and a DNA template into
consideration, the DNA clamping activity might potentially affect the
RF-C/PCNA interaction. However, the stimulation of RF-C ATPase was
independent of the DNA clamping of PCNA, implying that the initial
contact between RF-C and PCNA seems to occur in the absence of DNA, or
the DNA binding of RF-C alone may have primary significance for the
complex formation.
Figure 5:
The mutation sites and their activities
to stimulate RF-C ATPase or pol DNA synthesis. Filled circles indicate locations of mutations. Arrowheads indicate the
end points of deletions. Symbols in each stimulatory activity represent
extents of the activity by mutant PCNAs. +++, more than
80% activity of the wild type; ++, between 80 and 50% of the
activity; +, between 50 and 20% of the activity; and -, less
than 20% of the activity, respectively.
If these
affected mutation sites are placed on the crystal structure of PCNA,
they are positioned in several specific structures. Lys and Lys
are in two
-sheets (
I1 and
C2, respectively; see Fig. 6) corresponding to the
intermolecular interface of the PCNA trimer(22) . It has been
suggested that the interface may be necessary to hold the trimeric
structure. However, since these mutant proteins behave as a native
trimer during gel filtration chromatography (data not shown), this does
not appear to be the explanation of why these mutants are defective.
Thus, the mutation effects may be due to some local structural changes
which influence the DNA clamping activity or the contact with pol
. Asp
, Lys
, and Asp
are in
the loop between sets of
-sheets. These loops are exposed on the
outside of the protein and we suggest that they provide a structure to
contact directly with pol
.
Figure 6:
Summary of PCNA regions necessary for the
stimulation of RF-C ATPase and pol DNA synthesis. Boxed
regions indicate the mutation sites where the stimulatory activity
of RF-C ATPase and pol
DNA synthesis were affected. The height of
each box demonstrates the extent of the effect on each activity by
mutations according the results in Fig. 5. Neighboring mutation
sites of a similar result are included in a same box to indicate that
they may compose a functional region. Ambiguity for deletion ends of
the terminal functional region is indicated by slant lines with amino acid positions. The internal borders of functional
region of each terminal deletions have not been obtained by this study
and are indicated by broken lines. Bars with
A1 to
I2 indicate the structure units of yeast
PCNA(22) .
Interestingly, D41A affects both RF-C ATPase
stimulation and pol DNA synthesis. To exclude the possibility
that the D41A mutation would change the protein structure, we analyzed
the circular dichroism profile of D41A. The signals of short wave range
(200-250 nm) which represent peptide secondary structure were
completely same between wild PCNA and D41A. This indicates that D41A
has the same structure as wild PCNA. Thus, Asp
is required
to interact with both RF-C and pol
, and this loop has two targets
for interaction. But it is unclear whether or not this amino acid is
able to interact with pol
and RF-C simultaneously.
The CDC44 gene in S. cerevisiae encodes the large subunit
of RF-C, and five independent suppressors of its cold sensitive
mutations were isolated. All of them were mapped to the PCNA gene (pol 30), suggesting that the 140-kDa subunit interacts
directly with PCNA(26) . ()These mutations occurred
at positions completely or partially buried in the structure and are
likely to cause changes in the structure of PCNA(22) . Although
these suppressor mutation sites are involved in interacting with RF-C,
they are physically close to sites which we have shown to be involved
in interacting with pol
, but not with RF-C. This discrepancy may
due to a difference of the assay system. In our system, interaction of
RF-C and PCNA is studied by a biochemical assay and only strongly
affected mutations may have been appeared. In the case of suppressor
mutation, such strong mutations might not be isolated, but rather,
mutations that only moderately affect the protein could be isolated,
for example, those which alter the partial molecular structure will be
selected. It is therefore likely that the amino acid residues involved
in maintaining the tertiary structure may contribute to interaction
with RF-C as well as pol
, but their effect may not be detected in
our biochemical assay.
Figure 7:
A model for the assembly of pol
complex. A monomer of PCNA is indicated at the top. The four
-helices with nine basic amino acids are shown at the internal
surface. The C-terminal stretch with acidic amino acids is projected
from the molecule to emphasize the interaction site with RF-C. Shaded areas indicate the 6 predicted sites which may interact
with RF-C and pol
. The trimer PCNA molecule with a ring structure
is shown (second row), and the internal positive charges
provide interaction surface with DNA duplex. In the presence of RF-C
and ATP, PCNA is transferred onto the template/primer and forms the
primer recognition complex (third row). The large subunit (140
kDa) at the center and four small subunits (40, 38, 37, 36.5 kDa) of
RF-C are indicated in the elliptic shape. Subsequently, pol
is
incorporated and a highly processive DNA polymerase complex is formed (forth row).
We have introduced 33 mutations into the human PCNA gene and
studied their effects on the functions of PCNA. Stimulatory effects on
RF-C ATPase activity and pol DNA synthesis activity were examined
and the results are summarized in Fig. 5and Fig. 6.
Amino acid substitution at residues Lys
, Lys
,
Lys
, Lys
, Lys
,
Arg
, Arg
, Arg
, and
Lys
in PCNA, which are located at the
-helices,
exhibited a limited stimulation of pol
activity. The
-helices provide an internal surface to clamp a DNA template
through their highly positive charges facing to the same side of each
-helix (Fig. 7). In addition to the internal surface, the
-sheets at the intermolecular interface and the exposed loops have
a significant role in interaction with pol
. The assay to
stimulate RF-C ATPase with mutant PCNAs demonstrates a strict
requirement for specific residues that are located at the loops
projecting to one side of the PCNA trimer. PCNA has 6 redundant units
in the trimeric structure, each of which may have surfaces to interact
with either pol
or RF-C, or both.
How do pol and RF-C
share the redundant structures of PCNA? RF-C consists of five subunits,
and their cDNA sequences have demonstrated a significant structural
redundancy among them(30, 31) . Thus, we propose a
model to assemble the processive DNA polymerase complex as described in Fig. 7. At least three pockets of the side loops may interact
with three subunits of RF-C specifically. Other structures, such as the
intermolecular interface, may also contribute to interactions with
RF-C, as suggested by the yeast genetics. This means that the five
subunits of RF-C may occupy five out of six units of PCNA trimer.
Interaction between RF-C and PCNA in the presence of ATP may induce a
conformational change in PCNA, which could then open one of the
interfaces and pass a DNA helix through the PCNA ring. Then pol
may be recruited onto the template DNA using one free unit of PCNA as a
contact site.
Our study with single amino acid mutations and deletion mutations in PCNA demonstrates the presence of multiple functional surfaces in the characteristic ring structure of PCNA. In addition, several loops projected from one specific side provides the orientation which is important for the function of PCNA as a tracking molecule.