structural and functional insight into proliferating cell nuclear antigen
TRANSCRIPT
A 2016⎪Vol. 26⎪No. 0
J. Microbiol. Biotechnol. (2016), 26(4), 637–647http://dx.doi.org/10.4014/jmb.1509.09051 Research Article jmbReview
Structural and Functional Insight into Proliferating Cell Nuclear AntigenSo Young Park1,2†, Mi Suk Jeong1†, Chang Woo Han1, Hak Sun Yu2, and Se Bok Jang1*
1Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 46241, Republic of Korea2Department of Parasitology, School of Medicine, Pusan National University, Yangsan 50612, Republic of Korea
Introduction
Proliferating cell nuclear antigen (PCNA) is a highly
conserved protein in archaebacteria and several eukaryotic
species [67]. Studies of all known PCNAs show that they
are conserved in amino acid sequences, functions, and
structures [5, 54, 68]. PCNA was initially reported as a
fundamental auxiliary protein for the processes of DNA
replication and repair, and it works as a DNA sliding
clamp; that is, it stabilizes the interaction with other
proteins through a sliding platform [14, 30]. The DNA
sliding clamps perform an essential function in DNA
metabolic processes, and are found in eukaryotes,
prokaryotes, and archaea [24, 31, 37, 71, 74, 83]. They are
ring-shaped six-domain proteins that encircle duplex DNA
and enable highly processive DNA replication by serving
as binding sites for DNA polymerases. As a DNA clamp,
PCNA acts as a scaffold protein that organizes various
components for DNA replication, chromatin remodeling,
DNA damage repair, and cell cycle progression [47, 55].
PCNA is a homotrimer ring and it forms in a head-to-tail
arrangement of three monomers [36, 47]. This ring-shaped
trimeric PCNA is loaded onto DNA. The inner surface of
the PCNA is formed by 12 positively charged α-helices that
interact with DNA, and the outer layer contains 54 β-sheets
and interdomain-connecting loops (IDCLs) for protein-
protein interactions [47, 82]. In addition, PCNA interacts
with several translesion synthesis (TLS) DNA polymerases
[55, 84] and participates in repair of DNA that has been
damaged by chemotherapy agents [78], thereby conferring
chemotherapy resistance. PCNA was initially discovered to
be an antigen to the autoimmune disease systemic lupus
erythematosus [46, 71]. The level of expression of PCNA
changes during cell cycles and is associated with cell
proliferation or transformation [4]. A great deal of work
was conducted to determine the roles of PCNA in DNA
replication and one of the functions identified was a sliding
clamp for DNA polymerase δ [54, 68]. Deregulation of
PCNA expression is a hallmark of many proliferative
diseases and PCNA acts as a general proliferative marker,
particularly in determining cancer prognosis [34, 65].
The homotrimeric complex of PCNA consists of three
Received: September 17, 2015
Revised: December 18, 2015
Accepted: December 23, 2015
First published online
December 23, 2015
*Corresponding author
Phone: +82-51-510-2523;
Fax: +82-51-581-2544;
E-mail: [email protected]
†These authors contributed
equally to this work.
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2016 by
The Korean Society for Microbiology
and Biotechnology
Proliferating cell nuclear antigen (PCNA) is a critical eukaryotic replication accessory factor
that supports DNA binding in DNA processing, such as DNA replication, repair, and
recombination. PCNA consists of three toroidal-shaped monomers that encircle double-
stranded DNA. The diverse functions of PCNA may be regulated by its interactions with
partner proteins. Many of the PCNA partner proteins generally have a conserved PCNA-
interacting peptide (PIP) motif, located at the N- or C- terminal region. The PIP motif forms a
310 helix that enters into the hydrophobic groove produced by an interdomain-connecting
loop, a central loop, and a C-terminal tail in the PCNA. Post-translational modification of
PCNA also plays a critical role in regulation of its function and binding partner proteins.
Structural and biochemical studies of PCNA-protein will be useful in designing therapeutic
agents, as well as estimating the outcome of anticancer drug development. This review
summarizes the characterization of eukaryotic PCNA in relation to the protein structures,
functions, and modifications, and interaction with proteins.
Keywords: PCNA, DNA, structure, function, interaction
638 Park et al.
J. Microbiol. Biotechnol.
domains; the amino-terminal domain, the IDCL, and the
carboxyl-terminal domain. The IDCL links the N- and C-
terminal domains and makes significant contributions to
the diverse cellular activity of PCNA [21]. Investigations of
PCNA have shown that the IDCL is a major interaction site
for various binding proteins [19], including polymerases
Polδ, p21, DNA-(cytosine-5) methyltransferase (MeCTr),
DNA ligase 1 (LIG1), flap endonuclease 1 (FEN1), cyclin-
dependent kinase 2 (CDK2), and cyclin D, etc. Whereas it is
known that many proteins and peptides bind to PCNA
through conserved motifs, the binding mechanisms
between PCNA and partner proteins are still unknown. A
large part of the PCNA-binding proteins comprises a
conserved motif referred to as the PCNA-interacting
peptide (PIP) box [24, 28, 49]. The IDCL of PCNA is the
binding site that contains the consensus PIP-box motif,
Q-X-X-(I/L/M)-X-X-(F/Y)-(F/Y) [32, 84]. The PIP-box has
I/L/M small hydrophobic residues, F/Y aromatic side
chains, and X residues. Recently, another PCNA-binding
motif termed the KA-box [39], K-A(A/L/I)-(A/L/Q)-x-x-
(L/V), has been identified. It is also introduced in many
PCNA-interacting proteins and is associated with the
canonical PIP-box [62, 87]. Polη, Polι, and Polκ have a
noncanonical PIP-box sequence [25]. Other binding regions
are placed in the N-terminal region, including the
interaction between α-helices of PCNA and cyclin D [52].
In addition, the region is in the C-terminal region, which is
important for the interaction with Polε, CDK2, replication
factor C (RFC), and growth-arrest and DNA-damage-
inducible protein 45 (Gadd45) [39, 52]. This review focuses
on the structural characterization and function of PCNA.
The information will be useful in designing therapeutic
agents, as well as estimating the outcome of anticancer
drug development.
Major Role of PCNA in the DNA Replication and
Repair System
PCNA is a crucial replication factor that binds with many
partner proteins included in DNA replication and damage
repair. In mammals, DNA replication is started by
phosphorylation of the origin recognition complex at the
site of origin [8, 67]. PCNA is a homolog of the β subunit of
the DNA polymerases in prokaryotes, known as the sliding
clamp for eukaryotic DNA polymerases δ/ε [54, 68]. It
interacts with the Polδ switch to facilitate extension of
Okazaki fragments [79]. PCNA functions as a clamp
platform for polymerases δ and ε as well as a variety of
proteins at the replication. It is loaded onto DNA junctions
by the action of a multiple clamp loader, RFC, which
couples with ATP hydrolysis to open and close the PCNA
ring during replication and repair [23, 41]. PCNA exists as
stable trimers that form a closed ring with a central hole,
which encircles the DNA (Fig. 1). It interacts with and
recruits FEN1 and LIG1, which are required for Okazaki
fragment processing (Fig. 2) [39]. The PCNA complex for
the Okazaki fragment maturation ensures the directivity of
replication, and PCNA is a crucial moderator of DNA
replication. During the DNA replication, synthesis over
damaged templates is accomplished by polymerases via
translesion synthesis (TLS) [56, 90]. Conserved TLS
polymerases identified in several species includes Y-family
polymerases η, ι, and κ, and Rev1 and Rev7 as well as B-
family polymerase ζ [7, 53]. Mono-ubiquitination of PCNA
Fig. 1. Structural features of PCNA.
(A) Front view of the three-dimensional structure of human PCNA in
complex with the p21 peptide (PDB ID, 1AXC). PCNA is a trimeric
molecule, with each monomer containing a PIP-box binding site
located between the C-terminus of the protein and the interdomain
connector loop (IDCL). The side view shows the location of the IDCL
on the front side of PCNA and the extended loops on the back side.
The N-terminal domain is colored in red, the C-terminal domain is
colored in blue, and the IDCL is colored in green. (B) The molecular
surface of PCNA showing its charge distribution. Negatively charged
residues are shown in red; positively charged residues are shown in
blue.
Structural and Functional Roles of PCNA 639
April 2016⎪Vol. 26⎪No. 4
stimulates the recruitment of TLS polymerase to the
replication fork [23, 63]. PCNA provides the central stool
on which TLS polymerase can interact to acquire access to
the replicative stalled at the lesion site and to perform their
roles in modulation of different lesion bypass processes.
TLS polymerase works individually or in pairs according
to the damage type. PCNA enhances polymerase activity,
resulting in up-regulated ability of nucleotide on the other
sites to the damaged template [53, 61].
PCNA plays an important role in the DNA damage
repair and DNA replication system [72]. The major
metabolic pathways for DNA repair systems implicate
nucleotide excision repair (NER), base excision repair
(BER), double-strand break repair, and mismatch repair
(MMR) [10, 39, 85]. DNA damage by certain chemicals and
UV-irradiation results in bulky lesions, which are then
repaired via the NER pathway. During this process, PCNA
binds to the endonuclease, XPG (xeroderma pigmentosum
complementation group G) and facilitates new DNA
fragment resynthesis, which occurs after reactions catalyzed
by XPG [20]. PCNA is specifically loaded at the major
cellular activity site of the XPG 3’-incision to the lesion for
repairing [39]. BER is responsible for replacing chemically
altered nucleotide bases in DNA and can operate in either
short- or long-patch modes. PCNA is associated with DNA
repair in long-patch mode, which involves a DNA
polymerase δ/ε-dependent mechanism. It has been observed
to bind with various BER proteins, such as AP-endonuclease
1 (APE1), AP-endonuclease 2 (APE2), uracil-DNA glycosylase
2 (UNG2), nth endonuclease III-like 1, methylpurine-DNA
glycosylase (MPG), human MutY homolog, and X-ray
repair cross-complementing protein 1 (XRCC1) [49]. It is
possible that PCNA functions as a bridge for BER proteins
and stimulates their activities and acts as a coordinator for
the repair process [67]. MMR amends misincorporated
bases, which can produce from small insertion/deletion
and polymerase error loops achieved during recombination
and replication. It also operates for the beginning stages of
damage recognition [74]. The repair machinery takes away
the error-comprising part of a freshly synthesized strand,
and repairs targets to the newly generated single-stranded
gap [47]. In MMR, PCNA is needed for repair synthesis and
the beginning stages of damage recognition. MMR needs to
discriminate between the original and newly synthesized
strand to function properly. Because PCNA is loaded onto
the DNA in the only possible orientation, facing the 3’-end
of the daughter strand, discrimination is possible. Indeed,
exonuclease excisions of incorrectly incorporated nucleotides
in the growing strand are carried out in the 5’-3’ direction.
PCNA interacts directly with Msh6 (MutS homolog 6),
Msh3 (MutS homolog 3), Mlh1 (MutL homolog 1), and
EXO1 (exonuclease 1). MLH1 possesses PIP-boxes, MSH3,
and MSH6 [65].
Post-Translational Modifications of PCNA
PCNA is modified by several post-translational modifications,
including ubiquitylation, sumoylation, acetylation, and
phosphorylation. PCNA was recently shown to be subject
to nitrosylation at specific cysteine residues for which the
biological significance remains to be determined [1, 26].
Post-translational modification of PCNA seems to be
important for the polymerase switch, with post-translational
modification by ubiquitin being the best known. Mono-
ubiquitiylation of PCNA at Lys164 is induced by Rad6 and
Rad18 in a DNA damage-dependent manner [29, 60, 64],
which serves as a signal for activation of the translesion
synthesis pathway [75]. It is achieved by consequent
movement of the ubiquitin-activating enzyme E1, specific
Fig. 2. DNA replication and repair.
(A) During DNA replication, replicative polymerases, including DNA
polymeraseδ (Polδ), are associated with PCNA and ensure progression
of the replication fork mediated by the MCM complex. (B) Upon
encountering a replication block such as DNA damage, PCNA
modified by ubiquitylation (Ub) plays a key role in recruiting
translesion synthesis DNA polymerases, including Polη, which
initiates damage bypass. PCNA exists as stable trimers that form a
closed ring with a hole in the center that encircles duplex DNA.
640 Park et al.
J. Microbiol. Biotechnol.
ubiquitin-conjugation enzyme E2 (which in humans might
be either Rad6A or Rad6B), and RING (really interesting
new gene) finger-containing E3 ubiquitin ligase (Rad18)
[26, 91]. Mono-ubiquitylation of PCNA directs a switch
between processive DNA polymerase and TLS DNA
polymerase and results in error-prone bypass replication
[47]. In addition, polyubiquitylation of PCNA at Lys164
and Lys63 needs the heterodimeric ubiquitin-conjugation
enzyme Ubc13-Mms2 and a specific RING-finger-containing
E3 ubiquitin ligase, Rad5 (in yeast) [75]. Rad5 promotes
PCNA polyubiquitylation via interactions with both
Ubc13-Mms2 and PCNA. In humans, Rad5 orthologs, SNF2
histone linker PHD RING helicase, helicase-like transcription
factor, and RING finger protein 8, have been found to be
catalyzed by Mm2-Ubc13-dependent polyubiquitylation of
PCNA [11, 76]. Another PCNA modification is sumoylation
on Lys164, and to a lesser portion, Lys127, by the E2 SUMO
conjugating enzyme Ubc9 combination with E3 SUMO
ligase Siz1. The crystal structures of mono-ubiquitylated,
polyubiquitylated, and sumolylated PCNA were solved
[17, 18] and they showed that the modified positions were
located on the backside of PCNA (Figs. 3A and 3B). PCNA
Fig. 3. PCNA modifications.
(A and B) Side view of SUMO- and ubiquitin-modified PCNA. The SUMO (orange) and ubiquitin (magenta) groups are located on the back of
PCNA (cyan). SUMO and ubiquitin are oriented differently relative to PCNA. Left, ribbon representation; middle, analogous space-filling
representation; right, molecular surface representation of the SUMO- and ubiquitin-modified PCNA. (C) Structure of a single SUMOPCNA subunit
superimposed with the structure of ubiquitin from UbiPCNA is shown. The PDB ID is 3PGE for the SUMO-modified PCNA and 3L10 for the
ubiquitin-modified PCNA.
Structural and Functional Roles of PCNA 641
April 2016⎪Vol. 26⎪No. 4
sumoylation shows inhibition effects on the interaction of
PIP-box protein and PCNA. However, it has not yet been
characterized in mammals. Modification of PCNA by
ubiquitin and SUMO modulates the function of its target
protein by modifying, creating, or blocking the binding
motif. Both SUMO and ubiquitin are targeted to Lys164
residues of PCNA, and these modifications control the
various functions of PCNA [64]. In order to understand the
difference and similarity between post-translationally
modified forms of PCNA, the structures of UbiPCNA andSUMOPCNA were superimposed (Fig. 3C). Although these
modification positions coincide, the modifier positions are
quite different in both structures. UbiPCNA and SUMOPCNA
structures revealed that the ubiquitin and SUMO moieties
are located on the back face of the PCNA ring and interact
with the loop of PCNA via its hydrophobic surface [73].
The SUMO moiety is a more radial position than the
ubiquitin. This difference in modifier position is probably
caused by the longer flexible linker at the C-terminus of
ubiquitin in comparison with that of SUMO [11, 17].
Recently, new modifications of PCNA have been reported.
Aspartic acid and glutamic acid residues of PCNA undergo
esterification: methyl esterification on several aspartic
acids and glutamic acids residues. Interestingly, PCNA
methylation is associated with breast cancer and believed
to be cancer-specific [27]. Use of 2D-PAGE and a specifically
generated antibody revealed that an acidic isoform of
PCNA, cancer-specific PCNA (csPCNA), is exclusively
expressed in malignant tissues, including breast cancer,
prostate cancer, and esophageal adenocarcinoma, but not
in normal cells [82]. The functional results of these
modifications are not clearly known. The methyl esterification
of PCNA is likely to induce conformational changes in the
structure of PCNA, and it may facilitate or disrupt the
interaction with partner proteins. Therefore, PCNA has the
potential for use in the development of new cancer markers
and targeting of csPCNA in cancer cells.
PCNA in Cell Cycle and Apoptosis
PCNA-interacting proteins play major roles in the
regulation of the cell cycle. PCNA itself is a cyclin [49] that
is highly up-regulated during the S-phase and it binds to
cyclin-CDK complexes [86] as well as the CDK inhibitor,
p21 [21]. These interactions produce a PCNA-p21/CDK-
cyclin quaternary complex that could be independent from
the DNA replication machinery [89]. In the regulation of
cell cycle, p21 modulates critically the function of PCNA
[16]. p21Cip1/Waf1 is the main mediator of growth arrest
induced by p53 in response to DNA damage [13, 38, 69].
Tumor suppressor protein p21 having the PIP-box
modulates cell cycle progression by directly binding to
PCNA through its C-terminal region [48].
PCNA is involved in the regulation of damage-induced
apoptosis and programed cell death. The physical
interaction of PCNA with Gadd45 and MyD118 (myeloid
cell differentiation protein) has also been shown. They
have similar domains that mediate interaction of PCNA,
leading to negative cell growth [65, 77]. ING1 (inhibitor of
growth 1) is a tumor suppressor protein that binds PCNA
through the site used by growth regulatory proteins [70].
Specifically, the p33ING1 isoform of ING1 includes a PIP
domain that binds with PCNA [59]. Therefore, PCNA can
function as a bridging molecule that targets proteins with
distinct roles in DNA-based processes [39].
The PCNA structure has been conserved during evolution.
Human, rat, mouse, and Drosophila melanogaster PCNAs are
highly conserved in primary sequences [80]. The sequences
of full-length rat and human PCNAs are conserved, with
the exception of four amino acids (Fig. 4A). Human PCNA,
which consists of 261 amino acid residues, includes a
central hole used for interaction with DNA (Fig. 5) [21]. It is
composed of an N-terminal domain (amino acids 1-117), a
flexible IDCL (amino acids 118–135) and a C-terminal
domain (amino acids 136-261) (Fig. 4B). Crystallographic
study has shown that PCNA is composed of a toroid shape
structure in a head-to-tail manner [58]. The crystal structure
of yeast PCNA was determined and followed by the
human PCNA-p21 complex structure [88]. The structures
obtained from analysis of yeast, human, archaeal, and
plant PCNAs revealed similarities for the DNA polymerase
III β subunit [21, 36, 43, 66]. The DNA polymerase III β
subunit forms a homodimer with a six-fold symmetrical
ring, wherein each monomer consists of three repeating
domains [33]. Similarly, PCNA also exhibits a six-fold
symmetrical ring that encircles DNA. In contrast to the
two-subunit structure of DNA polymerase III β rings, most
of PCNA proteins have homotrimeric rings composed of
three PCNA homologs (PCNA1, PCNA2, and PCNA3)
(Fig. 6A). Head-to-tail arrangement of the three monomers
(29 kDa in human) gives rise to two distinct faces, the back
and front (C-terminus) [47, 67]. Each PCNA monomer
consists of two domains connected with an extended β-
sheet across the IDCL (Fig. 4A). PCNA monomers bind to
antiparallel interactions, resulting in six-fold symmetry
[26]. The PCNA ring has an overall negative charge with a
positively charged inner surface owing to the existence of
Lys and Arg [21]. The positively charged inner surface is
642 Park et al.
J. Microbiol. Biotechnol.
formed by α-helices interacted with the negatively charged
DNA backbone and the outer surface is composed of β-
sheets [45]. The PCNA monomer belongs to the α/β
protein family, which contains four α-helices and a twisted
β-sheet composed of 18 antiparallel β-strands (Fig. 4B) [51].
The interaction with partner proteins occurs on the front
side of PCNA where the IDCL is located. The back side of
PCNA is the site for post-translational modifications and
Fig. 4. Domain and secondary structures of PCNAs.
(A) Secondary structures of hPCNA, mPCNA, DmPCNA, and rPCNA. The secondary structure is shown according to the hPCNA structure.
Alpha helices are shown as rectangles and β-sheets as arrows. Loops are shown as black lines. Residues that are identical between the four species
are indicated blue-shadded Box. Sequence alignment was performed by ClustalW and Jalview. (B) Schematic diagram showing domains of the
full-length PCNA.
Fig. 5. Structure of PCNA bound to DNA.
PCNA-DNA model derived from PDB ID 3K4X. DNA forms a ~40° angle with the central axis of PCNA.
Structural and Functional Roles of PCNA 643
April 2016⎪Vol. 26⎪No. 4
contains several loops [65, 71]. PCNA structures are known
to complex with DNA, proteins, and peptides [2, 6, 21, 57].
The complexes formed through binding of PCNA to DNA
and proteins/peptides have provided valuable insight into
the mechanism by which PCNA functions during DNA
processing.
PCNA Interaction with Partner Proteins
Interaction with PCNA-partner proteins is a key
regulatory role in various PCNA cellular functions. PCNA
interacts directly with many of the proteins involved in
various cellular processes [47]. Table 1 represents the major
PCNA-dependent activities and the respective PCNA-
binding proteins. The interaction sites of PCNA are shown
with its partner proteins in Fig. 6A. PCNA interacts with
partner proteins via the hydrophobic patch on its front-
facing side created by the IDCL (amino acids 118-135), the
central loop (amino acids 41-44), and the C-terminal tail
(amino acids 254-257) [15]. The ICDL is a major interaction
site for several proteins, including p21, Pol δ, MeCTr, DNA
ligase 1, and FEN1 [39]. The globular N-terminus including
α-helices is the interaction site with cyclin D, and the
extreme C-terminus is crucial for interactions with Polε,
RFC, CDK2, and Gadd45 [9]. Many PCNA-interacting
partners include a conserved PIP-box PCNA-binding motif,
Q-X-X-(L/M/I)-X-X-(F/Y)-(F/Y), and they are consisted of
hydrophobic and aromatic residues for interaction (Fig. 6C)
[83]. A general motif controlling PCNA-protein interactions
is marked in quite a few of the human proteins [47]. The
PIP motif forms a 310 helix that enters into the hydrophobic
groove in the PCNA (Fig. 6B) [2, 6]. Recent studies have
focused on PIP-box interaction and identification of an
additional modulation protein-protein interface. The crystal
structure of human PCNA-p21CIP1/WAF1 showed the typical
interaction of a PCNA-PIP box (PDB ID, 1AXC) [21].
Interestingly, the mutation of PCNA with extensive affinity
for PIP-box revealed the DNA replication defects and the
elevated plasticity of PCNA for partner protein affinities
[40]. Some PCNA-interacting proteins do not possess the
PIP-box sequence. Instead, a PCNA interaction motif, K-A-
(A/L/I)-(A/L/Q)-x-x-(L/V), mediates PCNA interactions
[39, 87]. However, some PCNA-binding proteins can
interact with PCNA independently of the classical PIP-box
or the KA-box through another binding site on PCNA [49].
For example, FEN1 utilizes the PIP-box motif and its
flanking sequences located at the extreme C-terminal tail,
as well as several additional contacts from its globular N-
terminal region, to interact with the C-terminus and IDCL
on PCNA [57]. Because a large number of PCNA partner
candidates by database search studies contain pathways
that are irrelevant to DNA replication, DNA repair, or
Fig. 6. Binding sites on PCNA interaction partners.
(A and B) PCNA interacts with protein partners through the frontal hydrophobic groove organized by the central loop (CL, amino acids 41-44,
colored purple), the C-terminal tail (CT, amino acids 254-257, colored magenta), and the interdomain-connecting loop (IDCL, amino acids 118-
135, colored orange). (C) PIP-box and p21 PIP-box motifs are shown.
644 Park et al.
J. Microbiol. Biotechnol.
chromatin assembly, the identification of these additional
bindings may announce new functions of PCNA [15].
Summary
PCNA plays crucial roles in DNA replication, DNA
repair, the cell cycle, and apoptosis, and it interacts
with many partner proteins to accomplish these roles.
Conversion of PCNA-binding proteins can be started by
phosphorylation, proteolysis, affinity competition, and
modification of PCNA by sumoylation and ubiquitylation.
PCNA function is controlled by interaction partners as well
as post-translational modifications of itself. Many PCNA
partner proteins include PIP-box motifs and their binding
modes are conserved. A hydrophobic groove at the front of
PCNA serves as a docking site for the consensus PIP-box
motifs. An interdomain-connecting loop on the front-facing
PCNA ring serves as a major interaction site. PCNA
expression relates to cell proliferation and it has diagnostic
value in many types of cancers. In addition, it is also a
target for cancer therapy and its inhibitors are currently
being developed as potential anticancer drugs. These
studies for the interaction with partner proteins, signaling
regulation, or trimer formation of PCNA can approach to
develop new therapeutic agents. Two types of PCNA-
targeting peptide agents have been reported and some
peptides disrupt protein interactions and others prevent
the phosphorylation of Tyr211. Functional activation of
PCNA may contribute to a favorable patient response and
provide a therapeutic tool to overcome the development of
resistance to other therapies. Structural and biochemical
studies of the PCNA protein may provide a model target
for designing therapeutic agents, as well as evaluating the
efficacy of anticancer drugs. Further studies are being
conducted to identify the complex structures in PCNA and
its partner proteins/compounds, as well as structure-based
regulation of PCNA.
Acknowledgments
This study was supported by the Basic Science Research
Program through the National Research Foundation of
Table 1. PCNA-binding proeteins.
Activities Proteins
DNA polymerases polη, polι, polλ, polδ, polβ, polε, polκ, polζ, Rev1
Flap-endonuclease FEN1
DNA ligase DNA ligase 1
Topoisomerase Topo Iiα
Poly (ADP-ribose) polymerase PARP-1
Helicases, ATPases Srs2, WRN, RECQ5, Mgs1, Rrm3
Protein kinase CDK2, EGF Receptor
Clamp loader Rfc1, Rfc3, Rfc4
Mismatch repair enzyme Msh3, Msh6, Mlh1, EXO1
Base excision repair enzymes NTH1, XRCC1, APE1, APE2, UNG2, MPG, Hmyh
Nucleotide excision repair enzyme XPG
Histone chaperone CAF-1
Histone acetyltransferase p300
Histone deacetyltransferase HDAC1
DNA methyltransferase DNMT1
Replication licensing factor Cdt1
Sister-chromatid cohesion factors Chl1, Eco1, Ctf18
Chromatin remodeling factor WSTF
Cell cycle regulators p21, p57, Cyclin D1
E3 ubiquitin ligases Rad5, Rad18
E2 SUMO-conjugating enzyme Ubc9
Apoptotic factor Gadd45, p53, ING1b
PIP-box seqence containing proteins indicated by a bold letter. The proteins are originated from mammals.
Structural and Functional Roles of PCNA 645
April 2016⎪Vol. 26⎪No. 4
Korea (NRF) funded by the Ministry of Education, Science
and Technology (2014-046706) to S.B.J. This study was also
supported by the Research Fund Program of the Research
Institute for Basic Sciences, Pusan National University,
Korea, 2013 (Project No. RIBS-PNU-2013-203).
References
1. Armstrong AA, Mohideen F, Lima CD. 2012. Recognition of
SUMO-modified PCNA requires tandem receptor motifs in
Srs2. Nature 483: 59-63.
2. Bowman GD, O’Donnell M, Kuriyan J. 2004. Structural
analysis of a eukaryotic sliding DNA clamp-clamp loader
complex. Nature 429: 724-730.
3. Bray CM, West CE. 2005. DNA repair mechanisms in plants:
crucial sensors and effectors for the maintenance of genome
integrity. New Phytol. 168: 511-528.
4. Bravo R, Fey SJ, Bellatin J, Larsen PM, Celis JE. 1981.
Identification of a nuclear polypeptide (“cyclin”) whose
relative proportion is sensitive to changes in the rate of cell
proliferation and to transformation. Prog. Clin. Biol. Res. 85:
235-248.
5. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H. 1987.
Cyclin/PCNA is the auxiliary protein of DNA polymerase-
δ. Nature 326: 511-528.
6. Bruning JB, Shamoo Y. 2004. Structural and thermodynamic
analysis of human PCNA with peptides derived from DNA
polymerase-delta p66 subunit and flap endonuclease-1.
Structure 12: 2209-2219.
7. Burgers PM, Koonin EV, Bruford E, Blanco L, Burtis KC,
Christman MF, et al. 2001. Eukaryotic DNA polymerases:
proposal for a revised nomenclature. J. Biol. Chem. 276:
43487-43490.
8. Burgers PM. 2009. Polymerase dynamics at the eukaryotic
DNA replication fork. J. Biol. Chem. 284: 4041-4045.
9. Castrec B, Rouillon C, Henneke G, Flament D, Querellou J,
Raffin JP. 2009. Binding to PCNA in euryarchaeal DNA
replication requires two PIP motifs for DNA polymerase D
and one PIP motif for DNA polymerase β. J. Mol. Biol. 394:
209-218.
10. Chen C, Merrill BJ, Lau PJ, Holm C, Kolodner RD. 1999.
Saccharomyces cerevisiae pol30 (proliferating cell nuclear
antigen) mutations impair replication fidelity and mismatch
repair. Mol. Cell. Biol. 19: 7801-7815.
11. Chang DJ, Cimprich KA. 2009. DNA damage tolerance:
when it’s OK to make mistakes. Nat. Chem. Biol. 5: 82-90.
12. Chapados BR, Hosfield DJ, Han S, Qiu J, Yelent B, Shen B,
Tainer JA. 2004. Structural basis for FEN-1 substrate specificity
and PCNA-mediated activation in DNA replication and
repair. Cell 116: 39-50.
13. Chen IT, Akamatsu M, Smith ML, Lung FD, Duba D, Roller
PP, et al. 1996. Characterization of p21Cip1/Waf1 peptide
domains required for cyclin E/Cdk2 and PCNA interaction.
Oncogene 12: 595-607.
14. Chen X, Patel TP, Simirskii VI, Duncan MK. 2008. PCNA
interacts with Prox1 and represses its transcriptional activity.
Mol. Vis. 14: 2076-2086.
15. De Biasio A, Blanco FJ. 2013. Proliferating cell nuclear antigen
structure and interactions: too many partners for one
dancer. Adv. Protein Chem. Struct. Biol. 91: 1-36.
16. Dotto GP. 2000. p21 WAF1/Cip1: more than a break to the
cell cycle? Biochim. Biophys. Acta 1471: M43-M56.
17. Freudenthal BD, Gakhar L, Ramaswamy S, Washington MT.
2010. Structure of monoubiquitinated PCNA and implications
for translesion synthesis and DNA polymerase exchange.
Nat. Struct. Mol. Biol. 17: 479-484.
18. Freudenthal BD, Brogie JE, Gakhar L, Kondratick CM,
Washington MT. 2011. Crystal structure of SUMO-modified
proliferating cell nuclear antigen. J. Mol. Biol. 406: 9-17.
19. Freudenthal BD, Gakhar L, Ramaswamy S, Washington MT.
2009. A charged residue at the subunit interface of PCNA
promotes trimer formation by destabilizing alternate subunit
interactions. Acta Crystallogr. D Biol. Crystallogr. 65: 560-566.
20. Gary R, Ludwig DL, Cornelius HL, MacInnes MA, Park MS.
1997. The DNA repair endonuclease XPG binds to proliferating
cell nuclear antigen (PCNA) and shares sequence elements
with the PCNA-binding regions of FEN-1 and cyclin-
dependent kinase inhibitor p21. J. Biol. Chem. 272: 24522-
24529.
21. Gulbis JM, Kelman Z, Hurwitz J, O’Donnell M, Kuriyan J.
1996. Structure of the C-terminal region of p21 WAF1/CIP1
complexed with human PCNA. Cell 87: 297-306.
22. Guzinska-Ustymowicsz K, Pryczynicz A, Kemona A,
Czyzewska J. 2009. Correlation between proliferation markers:
PCNA, Ki-67, MCM-2 and antiapoptotic protein Bcl-2 in
colorectal cancer. Anticancer Res. 29: 3049-3052.
23. Haracska L, Johnson RE, Unk I, Phillips B, Hurwitz J,
Prakash L, Prakash S. 2001. Physical and functional
interactions of human DNA polymerase η with PCNA. Mol.
Cell. Biol. 21: 7199-7206.
24. Hingorani MM, O’Donnell M. 2000. Sliding clamps: a
(tail)ored fit. Curr. Biol. 10: R25-R29.
25. Hishiki A, Hashimoto H, Hanafusa T, Kamei K, Ohashi E,
Shimizu T, et al. 2009. Structural basis for novel interactions
between human translesion synthesis polymerases and
proliferating cell nuclear antigen. J. Biol. Chem. 284: 10552-
10560.
26. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S.
2002. RAD6-dependent DNA repair is linked to modification
of PCNA by ubiquitin and SUMO. Nature 419: 135-141.
27. Hoelz DJ, Arnold RJ, Dobrolecki LE, Abdel-Aziz W, Loehrer
AP, Novotny MV, et al. 2006. The discovery of labile methyl
esters on proliferating cell nuclear antigen by MS/MS.
Proteomics 6: 4808-4816.
28. Jónsson ZO, Hindges R, Hübscher U. 1998. Regulation of
646 Park et al.
J. Microbiol. Biotechnol.
DNA replication and repair proteins through interaction
with the front side of proliferating cell nuclear antigen.
EMBO J. 17: 2412-2425.
29. Kannouche PL, Wing J, Lehmann AR. 2004. Interaction of
human DNA polymerase η with monoubiquitinated PCNA:
a possible mechanism for the polymerase switch in response
to DNA damage. Mol. Cell 14: 491-500.
30. Kelman Z, O'Donnell M. 1995. Structural and functional
similarities of prokaryotic and eukaryotic DNA polymerase
sliding clamps. Nucleic Acids Res. 23: 3613-3620.
31. Kelman Z. 1997. PCNA: structure, functions and interactions.
Oncogene 14: 629-640.
32. Ko R, Bennett SE. 2005. Physical and functional interaction
of human nuclear uracil-DNA glycosylase with proliferating
cell nuclear antigen. DNA Repair 4: 1421-1431.
33. Kong XP, Onrust R, O'Donnell M, Kuriyan J. 1992. Three-
dimensional structure of the β subunit of E. coli DNA
polymerase III holoenzyme: a sliding DNA clamp. Cell 69:
425-437.
34. Kontopidis G, Wu SY, Zheleva DI, Taylor P, McInnes C,
Lane DP, et al. 2005. Structural and biochemical studies of
human proliferating cell nuclear antigen complexes provide
a rationale for cyclin association and inhibitor design. Proc.
Natl. Acad. Sci. USA 102: 1871-1876.
35. Koundrioukoff S, Jónsson ZO, Hasan S, de Jong RN, van
der Vliet PC, Hottiger MO, Hübscher U. 2000. A direct
interaction between proliferating cell nuclear antigen (PCNA)
and Cdk2 targets PCNA-interacting proteins for phosphorylation.
J. Biol. Chem. 275: 22882-22887.
36. Krishna TS, Kong XP, Gary S, Burgers PM, Kuriyan J. 1994.
Crystal structure of the eukaryotic DNA polymerase
processivity factor PCNA. Cell 79: 1233-1243.
37. Lee SD, Alani E. 2006. Analysis of interactions between
mismatch repair initiation factors and the replication
processivity factor PCNA. J. Mol. Biol. 355: 175-184.
38. Luo Y, Hurwitz J, Massagué J. 1995. Cell-cycle inhibition by
independent CDK and PCNA binding domains in p21Cip1.
Nature 375: 159-161.
39. Maga G, Hübscher U. 2003. Proliferating cell nuclear antigen
(PCNA): a dancer with many partners. J. Cell Sci. 116: 3051-
3060.
40. Mailand N, Gibbs-Seymour I, Bekker-Jensen S. 2013. Regulation
of PCNA-protein interactions for genome stability. Nat. Rev.
Mol. Cell Biol. 14: 269-282.
41. Majka J, Burgers PM. 2004. The PCNA-RFC families of DNA
clamps and clamp loaders. Prog. Nucleic Acid Res. Mol. Biol.
78: 227-260.
42. Mathews MB, Bernstein RM, Franza BR, Garrels JI. 1984.
Identity of the proliferating cell nuclear antigen and cyclin.
Nature 309: 374- 376.
43. Matsumiya S, Ishino Y, Morikawa K. 2001. Crystal structure
of an archaeal DNA sliding clamp: proliferating cell nuclear
antigen from Pyrococcus furiosus. Protein Sci. 10: 17-23.
44. Matsumiya S, Ishino S, Ishino Y, Morikawa K. 2002. Physical
interaction between proliferating cell nuclear antigen and
replication factor C from Pyrococcus furiosus. Genes Cells 7:
911-922.
45. McNally R, Bowman GD, Goedken ER, O'Donnell M,
Kuriyan J. 2010. Analysis of the role of PCNA-DNA contacts
during clamp loading. BMC Struct. Biol. 10: 3.
46. Miyachi K, Fritzler MJ, Tan EM. 1978. Autoantibody to a
nuclear antigen in proliferating cells. J. Immunol. 121: 2228-
2234.
47. Moldovan GL, Pfander B, Jentsch S. 2007. PCNA, the
maestro of the replication fork. Cell 129: 665-679.
48. Moskowitz NK, Borao FJ, Dardashti O, Cohen HD, Germino
FJ. 1995. The amino terminus of Cdk2 binds p21. Oncol. Res.
8: 343-352.
49. Naryzhny SN. 2008. Proliferating cell nuclear antigen: a
proteomics view. Cell. Mol. Life Sci. 65: 3789-3808.
50. Ollivierre JN, Silva MC, Sefcikova J, Beuning PJ. 2011.
Polymerase switching in response to DNA damage, pp. 241-
292. In Williams MC, Maher III LJ (eds.). Biophysics of DNA-
Protein Interactions: From Single Molecules to Biological
Systems. Springer-Verlag, Berlin.
51. Pan M, Kelman L, Kelman Z. 2011. The archaeal PCNA
proteins. Biochem. Soc. Trans. 39: 20.
52. Parsons JL, Nicolay NH, Sharma RA. 2013. Biological and
therapeutic relevance of nonreplicative DNA polymerases to
cancer. Antioxid. Redox Signal. 18: 851-873.
53. Prakash S, Johnson RE, Prakash L. 2005. Eukaryotic translesion
synthesis DNA polymerases: specificity of structure and
function. Annu. Rev. Biochem. 74: 317-353.
54. Prelich G, Tan CK, Kostura M, Mathews MB, So AG,
Downey KM, Stillman B. 1987. Functional identity of
proliferating cell nuclear antigen and a DNA polymerase-δ
auxiliary protein. Nature 326: 517-520.
55. Punchihewa C, Inoue A, Hishiki A, Fujikawa Y, Connelly
M, Evison B, et al. 2012. Identification of small molecule
proliferating cell nuclear antigen (PCNA) inhibitor that
disrupts interactions with PIP-box proteins and inhibits
DNA replication. J. Biol. Chem. 287: 14289-14300.
56. Riva F, Savio M, Cazzalini O, Stivala LA, Scovassi IA, Cox
LS, et al. 2004. Distinct pools of proliferating cell nuclear
antigen associated to DNA replication sites interact with the
p125 subunit of DNA polymerase δ or DNA ligase I. Exp.
Cell Res. 293: 357-367.
57. Sakurai S, Kitano K, Yamaguchi H, Hamada K, Okada K,
Fukuda K, et al. 2005. Structural basis for recruitment of
human flap endonuclease 1 to PCNA. EMBO J. 24: 683-693.
58. Schurtenberger P, Egelhaaf SU, Hindges R, Maga G, Majka
ZO, May RP, et al. 1998. The solution structure of
functionally active human proliferating cell nuclear antigen
determined by small-angle neutron scattering. J. Mol. Biol.
275: 123-132.
59. Scott M, Bonnefin P, Vieyra D, Boisvert FM, Young D,
Structural and Functional Roles of PCNA 647
April 2016⎪Vol. 26⎪No. 4
Bazett-Jones DP, Riabowol K. 2001. UV-induced binding of
ING1 to PCNA regulates the induction of apoptosis. J. Cell
Sci. 114: 3455-3462.
60. Sharma NM, Kochenova OV, Shcherbakova PV. 2011. The
non-canonical protein binding site at the monomer-monomer
interface of yeast proliferating cell nuclear antigen (PCNA)
regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent
translesion DNA synthesis. J. Biol. Chem. 286: 33557-33566.
61. Shcherbakova PV, Fijalkowska IJ. 2006. Translesion synthesis
DNA polymerases and control of genome stability. Front.
Biosci. 11: 2496-2517.
62. Shimazaki N, Yazaki T, Kubota T, Sato A, Nakamura A,
Kurei S, et al. 2005. DNA polymerase lambda directly binds
to proliferating cell nuclear antigen through its confined C-
terminal region. Genes Cells 10: 705-715.
63. Soria G, Gottifredi V. 2010. PCNA-coupled p21 degradation
after DNA damage: the exception that confirms the rule?
DNA Repair 9: 358-364.
64. Stelter P, Ulrich HD. 2003. Control of spontaneous and
damage-induced mutagenesis by SUMO and ubiquitin
conjugation. Nature 425: 188-191.
65. Stoimenov I, Helleday T. 2009. PCNA on the crossroad of
cancer. Biochem. Soc. Trans. 37: 605-613.
66. Strzalka W, Oyama T, Tori K, Morikawa K. 2009. Crystal
structures of the Arabidopsis thaliana proliferating cell
nuclear antigen 1 and 2 proteins complexed with the human
p21 C-terminal segment. Protein Sci. 18: 1072-1080.
67. Strzalka W, Ziemienowicz A. 2011. Proliferating cell nuclear
antigen (PCNA): a key factor in DNA replication and cell
cycle regulation. Ann. Bot. 107: 1127-1140.
68. Tan CK, Castillo C, So AG, Downey KM. 1986. An auxiliary
protein for DNA polymerase-delta from fetal calf thymus. J.
Biol. Chem. 261: 12310-12316.
69. Terry LA, Boyd J, Alcorta D, Lyon T, Solomon G, Hannon
G, et al. 1996. Mutational analysis of the p21/WAF1/CIP1/
SDI1 coding region in human tumor cell lines. Mol. Carcinog.
16: 221-228.
70. Thakur S, Feng X, Qiao Shi Z, Ganapathy A, Kumar Mishra M,
Atadja P, et al. 2012. ING1 and 5-azacytidine act synergistically
to block breast cancer cell growth. PLoS One 7: e43671.
71. Tsurimoto T. 1999. PCNA binding proteins. Front. Biosci. 4:
D849-D858.
72. Tuteja N, Singh MB, Misra MK, Bhalla PL, Tuteja R. 2001.
Molecular mechanisms of DNA damage and repair: progress
in plants. Crit. Rev. Biochem. Mol. Biol. 36: 337-397.
73. Ulrich HD, Takahashi T. 2013. Readers of PCNA modifications.
Chromosoma 122: 259-274.
74. Ulrich HD. 2009. Regulating post-translational modifications
of the eukaryotic replication clamp PCNA. DNA Repair 8:
461-469.
75. Umar A, Buermeyer AB, Simon JA, Thomas DC, Clark AB,
Liskay RM, Kunkel TA. 1996. Requirement for PCNA in
DNA mismatch repair at a step preceding DNA resynthesis.
Cell 87: 65-73.
76. Unk I, Hajdú I, Fátyol K, Hurwitz J, Yoon JH, Prakash L, et
al. 2008. Human HLTF functions as a ubiquitin ligase for
proliferating cell nuclear antigen polyubiquitination. Proc.
Natl. Acad. Sci. USA 105: 3768-3773.
77. Vairapandi M, Azam N, Balliet AG, Hoffman B, Liebermann
DA. 2000. Characterization of MyD118, Gadd45, and PCNA
interacting domains: PCNA impedes MyD/Gadd mediated
negative growth control. J. Biol. Chem. 275: 16810-16819.
78. Waters LS, Minesinger BK, Wiltrout ME, D’Souza S, Woodruff
RV, Walker GC. 2009. Eukaryotic translesion polymerases
and their roles and regulation in DNA damage tolerance.
Microbiol. Mol. Biol. Rev. 73: 134-154.
79. Waga S, Stillman B. 1998. The DNA replication fork in
eukaryotic cells. Annu. Rev. Biochem. 67: 721-751.
80. Wang K, Shi Z, Zhang M, Cheng D. 2013. Structure of
PCNA from Drosophila melanogaster. Acta Crystallogr. Sect. F
Struct. Biol. Cryst. Commun. 69: 387-392.
81. Wang SC, Nakajima Y, Yu YL, Xia W, Chen CT, Yang CC, et
al. 2006. Tyrosine phosphorylation controls PCNA function
through protein stability. Nat. Cell Biol. 8: 1359-1368.
82. Wang SC. 2014. PCNA: a silent housekeeper or a potential
therapeutic target? Trends Pharmacol. Sci. 35: 178-186.
83. Warbrick E. 2000. The puzzle of PCNA’s many partners.
Bioessays 22: 997-1006.
84. Winter JA, Bunting KA. 2012. Rings in the extreme: PCNA
interactions and adaptations in the archaea. Archaea 2012: 1-9.
85. Wood RD, Mitchell M, Sgouros J, Lindahl T. 2001. Human
DNA repair genes. Science 291: 1284-1289.
86. Xiong Y, Zhang H, Beach D. 1992. D type cyclins associate
with multiple protein kinases and the DNA replication and
repair factor PCNA. Cell 71: 505-514.
87. Xu H, Zhang P, Liu L, Lee MY. 2001. A novel PCNA-
binding motif identified by the panning of a random
peptide display library. Biochemistry 40: 4512-4520.
88. Zheleva DI, Zhelev NZ, Fischer PM, Duff SV, Warbrick E,
Blake DG, Lane DP. 2000. A quantitative study of the in
vitro binding of the C-terminal domain of p21 to PCNA:
affinity, stoichiometry, and thermodynamics. Biochemistry
39: 7388-7397.
89. Zhang H, Xiong Y, Beach D. 1993. Proliferating cell nuclear
antigen and p21 are components of multiple cell cycle
kinase complexes. Mol. Biol. Cell 4: 897-906.
90. Zheng L, Shen B. 2011. Okazaki fragment maturation:
nucleases take centre stage. J. Mol. Cell Biol. 3: 23-30.
91. Zhu Q, Chang Y, Yang J, Wei Q. 2014. Post-translational
modifications of proliferating cell nuclear antigen: a key
signal integrator for DNA damage response (Review). Oncol.
Lett. 7: 1363-1369.