1 DNA Enzymology and Molecular Virology, Istituto di Genetica Molecolare,
IGM-CNR, National Research Council, via Abbiategrasso 207, I-27100 Pavia,
Italy
2 Institute of Veterinary Biochemistry and Molecular Biology, University of
Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich,
Switzerland
* Author for correspondence (e-mail: hubscher{at}vetbio.unizh.ch)
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Summary |
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Key words: PCNA, PCNA-interacting proteins
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Too many dancing partners for PCNA |
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Three identical PCNA monomers, each comprising two similar domains, are
joined in a head-to-tail arrangement to form a homotrimer. This is thus
composed of six repeating domains and exibits six-fold symmetry
(Krishna et al., 1994;
Schurtenberger et al., 1998
).
The resulting ring has two non-equivalent surfaces: an outside surface
composed of ß-sheets; and a layer of
-helices rich in basic
residues lining the inner side of the hole, which are positioned
perpendicularly to the phosphate backbone of DNA. Owing to this unique
structure, PCNA is topologically linked to the double helix, encircling it,
but it is still able to freely slide along the DNA lattice by virtue of the
-helices lining the inner channel. Thus, PCNA and its homologs increase
the processivity of a polymerase by engaging in protein-protein interactions
with its outer surface and tethering it to the DNA. This property of PCNA
prevents the polymerase from dissociating while advancing along the template
DNA and is the reason for the name sliding clamp
(Kelman and O'Donnell,
1995
).
The binding sites on PCNA for many of its partners have been mapped
(Fig. 1) (reviewed by
Jonsson and Hubscher, 1997;
Warbrick, 2000
). A major
interaction site is the interdomain connecting loop, a coiled structure at the
side of PCNA, spanning residues from L121 to E132. This loop is recognised by
several proteins, such as pol
, p21, flap endonuclease 1 (Fen1), DNA
(cytosine) methyltransferase (MeCTr) and DNA ligase 1 (Lig1). Other important
sequences are the N-terminal region comprising the inner
-helices,
which forms part of the binding site for cyclin D, and the C-terminal tail,
which is important for the interaction with pol
, replication factor C
(RF-C), CDK2 and GADD45.
|
Many PCNA-binding proteins contain a common PCNA-binding motif: the PIP-box
(Jonsson et al., 1998;
Warbrick, 2000
), which has the
consensus sequence Q-xx-(h)-x-x-(a)-(a), where h represents residues with
moderately hydrophobic side chains (e.g. L, I, M), a represents residues with
highly hydrophobic, aromatic side chains (e.g. F, Y) and x is any residue.
Recently, by exploiting a random peptide display library, Xu et al. identified
a novel PCNA binding motif, K-A-(A/L/I)-(A/L/Q)-x-x-(L/V), termed the KA-box.
It is distinct from the `classical' PIP-box, and is also present in several
PCNA interacting proteins (Xu et al.,
2001
).
Tables 1 and
2 summarise PCNA-interacting
proteins and indicate their functions in the cell. These proteins can be
divided into two groups: those that have a known enzymatic activity
(Table 1) and those that are
involved in cell-cycle progression, checkpoint control or cellular
differentiation (Table 2). The
discovery of the overlapping nature of the binding sites for these PCNA
binding proteins immediately suggested that the different partners must bind
and dissociate sequentially to perform their functions
(Jonsson et al., 1998;
Warbrick, 2000
). Below we
provide examples of the importance of PCNA interactions in different DNA
metabolic pathways.
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PCNA in DNA replication |
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Synthesis of an Okazaki fragment is terminated when the pol or pol
holoenzyme meets the 5' end of the RNA portion of the previously
synthesised fragment and performs strand displacement synthesis. Finally,
specialised proteins are recruited that remove the RNA part, fill the gap and
ligate the two adjacent fragments. The two PCNA-binding proteins Fen1 and Lig1
are involved in this process (Bae and Seo,
2000
; Levin et al.,
2000
; Bae et al.,
2001
). Moreover, PCNA has been shown to stimulate Fen1 activity
(Jonsson et al., 1998
;
Tom et al., 2000
). In vitro
reconstitution of the Okazaki fragment maturation process showed that
competition for PCNA binding among pol
, Fen1 and Lig1 coordinates the
ordered action of these enzymes (Yuzhakov
et al., 1999
; Maga et al.,
2001
). When the pol
holoenzyme encounters the 5' end
of the previous fragment, it performs strand displacement synthesis in
conjunction with the helicase/endonuclease Dna2. The displaced strand
generates a flap structure, which is bound by the ssDNA-binding protein RP-A,
which, in turn, triggers dissociation of pol
from PCNA. Following the
recruitment of Fen1, the PCNA-Fen1 complex efficiently removes the flap,
leaving a nick in the double-stranded DNA. This is followed by binding of Lig1
to PCNA, which performs the final ligation step, thus sealing the nick
(Ayyagari et al., 2003
;
Jin et al., 2003
).
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PCNA in DNA repair |
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In vitro reconstitution of NER showed a requirement for PCNA
(Shivji et al., 1992), which
is probably due to the involvement of pol
and/or pol
in the
resynthesis step (Aboussekhra et al.,
1995
). The observation that PCNA also binds to the endonuclease
XP-G, which acts at a stage preceding DNA synthesis, suggested an additional
role for PCNA (Gary et al.,
1997
). Indeed, PCNA is loaded specifically at the site of the XP-G
incision, 3' of the lesion to be repaired. A protein important for the
damage-recognition step in the NER pathway is XP-A. Immunofluorescence studies
on wild-type or XP-A mutant cells showed that, upon UV irradiation, the
nuclear pattern and localisation of PCNA at the sites of DNA damage are
influenced by the presence or absence of a functional XP-A protein, suggesting
a role for PCNA in the early steps of the NER process
(Aboussekhra and Wood, 1995
;
Li et al., 1996
;
Miura and Sasaki, 1996
). Thus,
PCNA might help to recruit the essential proteins XP-G and XP-A to the site of
the lesion. After the incision has been made, PCNA can promote the transition
to the next steps by binding pol
(or pol
) for re-synthesis of
the complementary strand and subsequently recruiting Lig1 for the final
ligation step.
PCNA also functions in a step preceding DNA synthesis in MMR. Early studies
showed that PCNA interacts with mispair-binding proteins MSH2, MSH3 and MSH6
and enhances their mispair binding specificity
(Clark et al., 2000;
Flores-Rozas et al., 2000
;
Kleczkowska et al., 2001
).
Recently, Lau and Kolodner have shown that PCNA and MSH2-MSH6 form a stable
ternary complex on newly replicated DNA and that this complex is transferred
from PCNA to mispaired bases in an ATP-dependent fashion
(Lau and Kolodner, 2003
).
Thus, PCNA has also a role in recruiting and coordinating repair proteins in
the MMR pathway.
PCNA similarly functions in long-patch BER in vitro, where the DNA
synthesis step is dependent upon pol and/or pol
(Klungland and Lindahl, 1997
;
Fortini et al., 1998
;
Gary et al., 1999
;
Pascucci et al., 1999
;
Levin et al., 2000
;
Matsumoto, 2001
). Moreover,
Matsumoto et al. have identified a PCNA-dependent pathway in Xenopus
laevis for repairing abasic sites, which is an alternative to the pol
ß-dependent mechanism (Matsumoto et
al., 1994
; Matsumoto et al.,
1999
). This reaction depends on AP endonuclease, Fen1, RF-C and
pol
. The observation that PCNA physically interacts with the AP
endonucleases Apn and Apn2, and the uracil DNA glycosylase UNG
(Dianova et al., 2001
;
Krokan et al., 2001
;
Unk et al., 2002
) supports the
notion that PCNA participates both in the incision and in the resynthesis
steps of BER.
A recent observation provided a link between PCNA and the
post-replicational RAD6-dependent repair pathway
(Hoege et al., 2002).
Essential elements of this pathway are the ubiquitin-conjugating enzymes RAD6
and the MMS2-UBC13 heterodimer, the RING-finger proteins RAD18 and RAD5, and
the small ubiquitin-related modifier (SUMO)-conjugating enzyme UBC9. PCNA is
mono-ubiquitylated by RAD6 and RAD18, multi-ubiquitylated in an MMS2, UBC13
and RAD5-dependent manner, and conjugated to SUMO by UBC9
(Hoege et al., 2002
). These
modifications affect a specific residue (K63) of PCNA and they are essential
for damage-induced DNA repair, differentially affecting resistance to DNA
damage.
An alternative pathway for DNA damage tolerance in eukaryotic cells is
translesion synthesis (TLS), which relies on specialised polymerases able to
carry on DNA synthesis on a damaged template
(Goodman, 2002;
Hubscher et al., 2002
). This
pathway plays a major role in S phase, where replication forks can stall at
damaged sites, owing to the inability of replicative pols to replicate damaged
DNA. Replication fork stalling can lead to DSBs, which trigger S phase
checkpoint mechanisms leading either to recombinational repair, which gives an
increased incidence of deletion/duplication and chromosomal alteration, or to
apoptosis. In order to avoid such dramatic consequences, cells can replace
replicative polymerase at stalled forks with specialised TLS polymerases (see
hypothetical model in Fig. 2)
that can incorporate a nucleotide in front of the lesion, and thus create a
3'-OH primer that can be extended by replicative polymerases. Examples
of these TLS enzymes are pol
, pol
, pol
and pol
(Hubscher et al., 2002
).
Recent results have shown that the lesion bypass activity of these enzymes is
increased by physical interaction of PCNA
(Haracska et al., 2001a
;
Haracska et al., 2001b
;
Haracska et al., 2002
;
Maga et al., 2002
), and
immunofluorescence studies have shown that pol
and pol
accumulate at stalled replication forks following DNA damage
(Kannouche et al., 2002
).
Since TLS requires interplay between these specialised polymerases and the
replicative pol
and pol
, PCNA appears to be the ideal candidate
for coordinating their functions, as well as for recruiting them at the
replication fork (Fig. 2).
|
A mutation in the gene encoding pol has been shown to be associated
with the variant form of the genetic disease Xeroderma Pigmentosum
(XP-V) (Masutani et al.,
1999
). Pol
binds to PCNA
(Haracska et al., 2001b
), as
does the product of another disease-linked gene, the RecQ-like DNA helicase
WRN. Mutations in WRN cause increased genetic instability, leading to the
disease Werner Syndrome. WRN is involved in the recombinational repair of
double-strand breaks and has been shown to interact with PCNA through its
N-terminal domain (Lebel et al.,
1999
). Moreover, WRN also physically interacts with pol
and Fen1, two other PCNA-binding proteins, stimulating their catalytic
activities (Kamath-Loeb et al.,
2000
; Brosh et al.,
2001
; Kamath-Loeb et al.,
2001
). The role of PCNA in the recombinational repair pathway
might thus depend on the WRN helicase.
Given the central role of PCNA in almost all DNA repair pathways, an
intriguing scenario in which PCNA is bound to the DNA and works as general
recruiting agent for specific factors can be envisaged. Depending on the kind
of damage, the chromosomal context and the particular moment of the cell
cycle, PCNA might help to create an optimal `window' for the action of a
particular repair pathway, allowing access to the lesioned DNA only to a
specific set of proteins. Indeed, PCNA been shown to facilitate the catalytic
turnover of repair-specific enzymes involved in the incision step of NER,
presumably through interaction with repair-specific proteins
(Nichols and Sancar, 1992).
Moreover, the tight association of p21 (CIP1/WAF1) with PCNA in the nucleus
following DNA damage (Li et al.,
1996
) suggests that p21, together with PCNA, can link DNA
replication, DNA repair and cell-cycle progression. For example, upon DNA
damage, p21 may bind to PCNA and this may contribute to the XP-A and
XP-G-dependent damage-recognition and incision steps. If the repair machinery
fails to repair the lesions promptly, p21 and PCNA may continue to accumulate
at those damaged sites, recruiting alternative factors, which, in turn,
determine the cell's fate.
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PCNA in cell cycle control |
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Biochemical studies showing that PCNA interacts with the S-phase-specific
CDK2cyclin-A complex, suggest a functional role for binding of PCNA to
cyclin-CDK complexes (Koundrioukoff et
al., 2000). Physical binding of CDK2 to the C-terminal region of
the PCNA trimer produces an active ternary PCNACDK2cyclin-A
complex. PCNA appears to act as a connector between CDK2 and its substrates
(e.g. RF-C and Lig1), stimulating their phosphorylation. PCNA might thus
target the CDK2cyclin-A complex to PCNA-binding DNA replication
proteins. This might represent an important regulatory mechanism for the
recruitment of specific proteins to sites of DNA replication. For example, in
mammalian cells two immunologically distinct pol-
-primase
subpopulations have been identified (Dehde
et al., 2001
), which differ in the phosphorylation status of the
p68 regulatory subunit of pol
. This post-translational modification is
catalysed by the CDK2cyclin-A complex. By using monoclonal antibodies
selective for the two enzyme populations it was shown that only the
phosphorylated enzyme co-immunoprecipitates with cyclin A and colocalizes with
replication factories.
DNA damage, senescence or differentiation of cells through either
p53-dependent or -independent pathways induces the expression of the p21
protein, which blocks progression from G1 to S phase of the cell cycle. p21
binds to CDKs through its N-terminal region and to PCNA through its C-terminal
region (Chen et al., 1995;
Luo et al., 1995
;
Chen et al., 1996
;
Moskowitz et al., 1996
). In
vitro binding of p21 to PCNA results in inhibition of DNA replication but not
of PCNA-dependent NER. Biochemical analyses showed that p21 efficiently
inhibits DNA elongation during processive synthesis by pol
(or pol
) but not during the short gap-filling activity required in NER
reactions (Podust et al.,
1995
; Shivji et al.,
1998
). These results suggest that PCNA is an essential mediator of
the regulatory action of p21 (Waga et al.,
1994c
). Recent results have shown that, in terminally
differentiated cardiomyocytes, cell cycle arrest is dependent on the
maintainance of high levels of p21, which, in turn, are required for the
downregulation of PCNA (Engel et al.,
2003
). One possible explanation is that p21 prevents PCNA from
binding to the cell cycle or DNA replication machinery, thus targeting the
protein for destruction. Indeed, p21 can form a stable complex with PCNA on
DNA, preventing further interaction with the replication proteins RF-C and pol
(Waga et al.,
1994b
).
Together, these observations allow us to envisage a possible scenario in which PCNA is both a transducer and a target of positive and negative signals. Binding of PCNA to cyclin-CDK complexes might help to bring these regulatory proteins to their targets, whereas disruption of these interactions by the competitive binding of p21 is a signal for DNA replication arrest.
DNA replication proteins appear to be organised in large macromolecular
complexes or `factories', which in S-phase colocalise with the sites of newly
synthesized DNA (Hozak et al.,
1993; Hozak et al.,
1994
; Jackson,
1995
). It has been shown that the PCNA-binding domain located in
the N-terminal half of Lig1 and RF-C is necessary and sufficient to target
these proteins to replication foci
(Montecucco et al., 1998
).
Moreover, association of Lig1 with PCNA as well as its recruitment to
replication factories is regulated by phosphorylation in a
cell-cycle-dependent fashion (Rossi et
al., 1999
). These results suggested that PCNA acts as a recruiting
center for DNA replication proteins, helping to build the dynamic replisome at
the replication fork.
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PCNA and its partners in other cellular events |
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PCNA and gene expression
The methylated CpG sequences of the mammalian genome are heritable and
affect gene expression. The enzyme responsible for inheritance of the
methylated status is MeCTr. PCNA can bind to MeCTr, which favours the idea
that maintenance of the methylation pattern in the genome is also dependent on
PCNA (Chuang et al., 1997;
Iida et al., 2002
).
PCNA and sister chromatin cohesion
Sister chromatin cohesion is essential for the coordinated separation of
replicated chromosomes into daughter cells during mitosis. A component of the
cohesion complex called Ctf7p (for chromosomal transmission fidelity) links
mitotic chromosome structure to the DNA replication machinery, since it is
only required during S phase. This connection might involve PCNA since it
genetically interacts with Ctf7p (Skibbens
et al., 1999). A proteomic approach to identify PCNA-binding
proteins in human cell lysates identified another cohesion factor that can
bind PCNA, CHL12 (Ohta et al.,
2002
). The fact that CHL12 can in addition bind to the four small
subunits of RF-C (see above) suggests that it might act as an alternative
clamp loader for PCNA and further indicates a connection between chromatid
cohesion and DNA replication.
PCNA and apoptosis
It has been known for some years that both the growth
arrest and DNA damage gene product Gadd45 and
the myeloid differentiation primary response gene
product MyD118 can physically interact with PCNA (reviewed by
Jonsson and Hubscher, 1997).
More recently, Vairapandi et al. found that similar domains in Gadd45 and
MyD188 mediate the interaction with PCNA
(Vairapandi et al., 2000
).
When mutants of Gadd45 or MyD188 that lack the PCNA-interaction domain are
ectopically expressed, they induce apoptosis more efficiently. This led to the
conclusion that the interaction of Gadd45 and MyD188 with PCNA triggers
negative growth control.
ING1 is another protein that might link PCNA to inhibition of cell growth
and/or apoptosis. It is a potential tumour suppressor and the isoform
p33ING1 contains a PIP motif and physically interacts with PCNA
(Scott et al., 2001). Cells
expressing ING1 mutants that cannot bind to PCNA are protected from UV-induced
apoptosis.
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Conclusions and perspectives |
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How are these interactions co-ordinated?
Table 3 summarises the known
affinities of different partners for PCNA. All are in the nanomolar range,
even if there are some differences. Pol , pol
, RF-C and p21 show
the strongest interactions, whereas Fen1 and Lig1 bind with somewhat lower
affinity to PCNA. This suggest that p21 could be an effective competitor of
Fen1 and Lig1 binding, which is consistent with biochemical studies. However,
the generally similar affinities for PCNA of its partners indicate that some
additional level of regulation must exist. One possible mechanism, supported
by experimental data, is regulation of the levels and the localisation of the
different PCNA-interacting proteins. This is well exemplified by the dynamics
of p21 and PCNA during the cell cycle. In normal quiescent fibroblast, p21 and
PCNA are present in the cell at an almost equimolar ratio but show different
distributions in the nucleus (Li et al.,
1996
). Upon entering in S phase, PCNA levels rise, whereas p21
levels remain low. This allows PCNA to bind other proteins without being
challenged by p21, and indeed several replication proteins colocalise in
discrete foci with PCNA during S phase. Upon DNA damage, by contrast, p21
levels increase dramatically and p21 colocalises at PCNA foci, which is
consistent with the idea that it can displace other partners from PCNA by
virtue of its high binding affinity. Similarly, Chuang et al. have shown that
the relative levels of p21 and MeCTr, another partner competing for PCNA
binding, are correlate with the proliferative state of the cells. In normal
cells, p21 levels are higher than MeCTr levels whereas, in SV40-transformed
cells, MeCTr is in excess over p21. Accordingly, PCNA can be
co-immunoprecipitated with MeCTr only from transformed cells. Upon DNA damage,
p21 colocalises with PCNA at repair sites and can effectively prevent
methylation of damaged DNA by precluding MeCTr interaction with PCNA, but only
in non-transformed cells (Chuang et al.,
1997
). Thus, it is clear that expression levels as well as
cellular localisation play an important role in regulating the balance between
PCNA-interacting proteins.
|
Another possible mechanism of regulation might lie in the homotrimeric
structure of PCNA which, in principle, can allow it to bind different partners
simultaneously. There is some indirect evidence suggesting that this can
happen. For example, p21 has been shown to bind to PCNA with a 3:1
stoichiometry, which is consistent with the notion that it prevents other
interactions by occupying all the possible binding sites on the trimer
(Gulbis et al., 1996).
Biochemical studies suggested that another interacting partner, Gadd45, binds
to PCNA with a 2:1 stoichiometry, thus leaving a binding site free for other
proteins to interact (Hall et al.,
1995
). Also, PCNA can have distinct binding modes. For example, in
the absence of DNA, Fen1 and Apn2 interact with PCNA mainly through the
interdomain-connecting loop; however, when PCNA encircles the DNA, the
C-terminal domain of PCNA becomes more important for binding of the two
partners (Gomes and Burgers,
2000
; Unk et al.,
2002
). The switch between alternative binding sites might be
another way in which PCNA can regulate its interaction with different
partners.
Very recently, experiments with the heterotrimeric PCNA from the
hyperthermophylic archeon Sulfolobus solfataricus suggested that one
subunit of the trimer interacts with the archeal polymerase, whereas the other
two subunits interact with the S. solfataricus homologs of Lig1 and
Fen1, respectively (Dionne et al.,
2003).
Post-translational modifications of both PCNA and its binding proteins, such as acetylation, SUMOylation or phosphorylation, can also positively or negatively regulate the interaction. We have now a profile of the PCNA partners. What is needed is a more mechanistic view of the processes: we must determine binding constants, the order of activation and other biochemical parameters. This will enable us to understand the mechanisms that spatially and temporally regulate the ability of PCNA to `dance' with the right partner at the right time.
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Acknowledgments |
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