Proliferating cell nuclear antigen
The 5'-->3'-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5'-->3'-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions (Li, 1995).
Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene
products specifically participate in different DNA transactions are not well understood. Fractionation of a
HeLa nuclear extract by DNA ligase I affinity chromatography results in the specific retention of a replication protein,
proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments have demonstrated that DNA ligase I
and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is
required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around
duplex DNA, interacts with DNA pol delta and enables this enzyme to synthesize DNA processively. An interaction
between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibits
PCNA-dependent DNA synthesis by DNA pol delta. These observations suggest that a ternary complex of DNA
ligase I, PCNA and DNA pol delta does not form on a gapped DNA template. Consistent with this idea, the cell cycle
inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol delta, disrupts the
DNA ligase I-PCNA complex. Thus, it is proposed that after Okazaki fragment DNA synthesis is completed by a
PCNA-DNA pol delta complex, DNA pol delta is released, allowing DNA ligase I to bind to PCNA at the nick
between adjacent Okazaki fragments and catalyze phosphodiester bond formation (Levin, 1997).
In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of
approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This
motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues.
N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating
cell nuclear antigen (PCNA) in vitro. Moreover, both the RFTS of DNA ligase I and of the RF-C large subunit are necessary
and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS
are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the
RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), it is
proposed that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the
recruitment and stable association of DNA replication proteins at replication factories (Montecucco, 1998).
The recruitment of DNA ligase I to replication foci in S phase depends on a replication
factory targeting sequence that also mediates the interaction with proliferating cell nuclear
antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a
phospho-epitope, it has been demonstrated that Ser66 of DNA ligase I, which is part of a strong
CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After
dephosphorylation in early G1, the level of Ser66 phosphorylation is minimal in G1, increases progressively in S and
peaks in G2/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of
Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. DNA
ligase I and PCNA interact in vivo in G1 and S phase but not in G2/M. It is proposed that dephosphorylation of Ser66 is
part of a novel control mechanism to establish the pre-replicative form of DNA ligase I (Rossi, 1999).
DNA polymerase delta is the functional
DNA polymerase on the leading strand of the eucaryotic DNA replication fork. A polyacrylamide gel electrophoresis band-mobility shift assay was developed to study the binding
of synthetic oligonucleotides by DNA polymerase delta and proliferating cell nuclear
antigen (PCNA). As measured by this assay, neither calf thymus pol delta core enzyme nor PCNA
alone bind DNA stably. However, mammalian PCNA (but not Drosophila PCNA) promotes the
formation of a distinct pol delta.PCNA template-primer complex. Appearance of this complex is
primer-dependent but does not require Mg2+. Complex stability is also influenced by the presence
or absence of individual dNTPs. This study proposes a model for the ordered sequential interaction of pol delta, PCNA,
and DNA template-primers (Ng, 1997).
The high speed and processivity of replicative DNA polymerases
reside in a processivity factor which has been shown to be a ring-shaped protein. This
protein, a so-called "sliding clamp," encircles DNA and tethers the catalytic unit to the template.
In eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity
factors are ring-shaped; despite this uniformity, they assume different oligomeric states. For example, the Escherichia coli
clamp (the beta subunit) is active as a dimer, while the eukaryotic and T4 phage
clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not
assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes
ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from
DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60
PM) and on DNA (t1/2 approximately 1 h) than PCNA, which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA, as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase
from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) are
also capable of unloading their respective clamps from DNA in the presence of ATP. It is concluded that the stability of the different clamps in solution correlates with their
stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta may double as clamp unloaders, presumably for the purpose of clamp recycling (Yao, 1996).
The remarkable processivity of cellular replicative DNA polymerases derive their tight
grip to DNA from a ring-shaped protein that encircles DNA and tethers the
polymerase to the chromosome. The crystal structures of prototypical "sliding clamps"
of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for
encircling DNA. Although beta is a dimer and PCNA is a trimer, their structures are
nearly superimposable. Even though they are not hexamers, the sliding clamps have a
pseudo 6-fold symmetry resulting from three globular domains comprising each beta
monomer, and two domains comprising each PCNA monomer. These domains have
the same chain fold and are nearly identical in three-dimensions. The amino acid
sequences of 11 beta and 13 PCNA proteins from different organisms have been
aligned and studied to gain further insight into the relation between the structure and
function of these sliding clamps. Furthermore, a putative embryonic form of PCNA is
the size of beta and thus may encircle DNA as a dimer, like the prokaryotic clamps (Kelman, 1995).
By site-directed mutagenesis and biochemical analyses, a study was performed of the functional domains of human PCNA required for stimulation of
replication factor C (RF-C) ATPase and DNA synthesis by pol delta. Short deletions
from either the N or C termini cause 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 Lys254 to
Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of
DNA synthesis by pol delta. Nine basic amino acids that are essential for activating
DNA synthesis by pol delta are positioned at the internal alpha-helices of PCNA. This
result is in good agreement with the observation that PCNA has a ring structure
similar to the bacterial beta-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 delta DNA synthesis. Some of them are positioned at
loops, exposed on one of the side surfaces of PCNA adjacent to the
C-terminal loop. In addition, the beta-sheets composing the intermolecular interface of
the trimeric PCNA are important for interaction with pol delta. Therefore, the outer
surface of PCNA has multiple functional surfaces that are responsible for the
interaction with multiple factors. The two side surfaces seem to be
functionally distinguishable, and this may determine the orientation of tracking PCNA
along the DNA (Fukuda, 1995).
Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are
processivity factors for eukaryotic DNA polymerases delta and epsilon. RFC carries out
multiple functions: these include the ability to recognize and bind to a DNA primer end and
load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then
tethers the polymerase to the template, allowing processive DNA chain elongation.
Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36); RFC
activity can be reconstituted from the five cloned gene products. To characterize the
role of the large subunit p140 in the function of the RFC complex, deletion mutants
were created that defined a region within the p140 C terminus required for complex
formation with the four small subunits. Deletion of the p140 N-terminal half, including
the DNA ligase homology domain, results in the formation of an RFC complex with
enhanced activity in replication and PCNA loading. Deletion of additional N-terminal
amino acids, including those constituting the RFC homology box II that is conserved
among all five RFC subunits, disrupts RFC replication function. DNA primer end
recognition and PCNA binding activities, located in the p140 C-terminal half, are
unaffected in this mutant, but PCNA loading is abolished (Uhlmann, 1997a).
Replication factor C subunits share regions of high homology, including the defined RFC boxes
II-VIII. RFC boxes III and V constitute a putative ATP binding site, whereas the
function of the other conserved boxes is unknown. To study the individual subunits in
the RFC complex and the role of the RFC boxes, deletion mutations were created in
all subunits. Sequences close to the C terminus of each of the small subunits are
required for formation of the five subunit complex. A N-terminal region of the small
subunits, containing the RFC homology box II, plays a critical role in the function of
these subunits. Deletion of the RFC homology box II reduces but does not abolish RFC activity in loading
PCNA onto DNA. The RFC homology box II also acts in supporting an RFC-dependent replication reaction.
The N termini of p37 and p40, although highly homologous, are not interchangeable,
suggesting unique functions for the individual subunits (Uhlmann, 1997b).
Replication factor C (RF-C) is a heteropentameric protein essential for DNA
replication and DNA repair. It is a molecular matchmaker required for loading of the
proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and
for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The
DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned. The PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. However, the DNA binding domain is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the
formation of an active sliding clamp by DNA polymerases delta and epsilon (Maga, 1997).
DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa
catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is
distributive by itself and requires an accessory protein, the proliferating cell nuclear
antigen (PCNA), for highly processive DNA synthesis. The catalytic subunit of human DNA polymerase delta (p125)
expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric
calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To
determine whether the lack of response to PCNA of the recombinant catalytic subunit
is due to the absence of the small subunit or to differences in post-translational
modification in insect cells versus mammalian cells, the two
subunits of human DNA polymerase delta were co-expressed in insect cells. Co-expression of the catalytic and small subunits of human DNA polymerase delta
results in formation of a stable, fully functional heterodimer. The recombinant
heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by
PCNA, and the increase in activity seen in the presence of PCNA is the result of
an increase in processivity. These data establish that the 50 kDa subunit is essential
for functional interaction of DNA polymerase delta with PCNA and for highly
processive DNA synthesis (Zhou, 1997).
A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after
nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus
DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers
containing a mispaired nucleotide at the 3'-terminal position of the primer. This
complex is indistinguishable in composition from that formed with a fully base paired
template-primer. Extension of a mispaired primer terminus is a component of DNA
polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide
template-primers was compared with and without its specific processivity factor,
PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide
for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions
reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to
incorporate (and/or not excise) the dTTP analog,
2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model
is proposed whereby the increased stability (decreased off-rate) of the pol
delta.template-primer complex in the presence of PCNA facilitates unfavorable
events catalyzed by pol delta. This model suggests an explicit mechanistic requirement
for the intrinsic 3'-5'-exonuclease of pol delta (Mozzherin, 1996).
The direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called
Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition
of PCNA-dependent DNA synthesis. p21 first inhibits the replication
factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA
polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most
to the inhibition of pol delta holoenzyme activity. p21 primarily inhibits the DNA synthesis resulting
from multiple reassembly of DNA polymerase delta holoenzyme. In contrast, the ability of the
PCNA clamp to translocate along double-stranded DNA is not affected by p21. These data were
confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibits clamp
assembly nor prevents the loading of DNA polymerase delta core onto DNA. These data suggest
that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template
length but does act preferentially on polymerization that encounters obstacles to progress (Podust, 1995).
The Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30
gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed
mutations were constructed in the POL30 gene, each mutation changing two adjacently located
charged amino acids to alanines. Although none of the mutant strains containing these double-alanine
mutations as the sole source of PCNA are temperature sensitive or cold sensitive for growth,
about a third of the mutants show sensitivity to UV light. Some of those UV-sensitive mutants have
elevated spontaneous mutation rates. In addition, several mutants suppress a cold-sensitive
mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A
cold-sensitive mutant, which was isolated by random mutagenesis, shows a terminal phenotype at
the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were
expressed and purified from Escherichia coli, and their in vitro properties determined. The
cold-sensitive mutant (pol30-52, S115P) is a monomer, rather than a trimer, in solution. This
mutant is deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta
holoenzyme activity is achieved at 37 degrees C but not at 14 degrees C by inclusion of the
macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6,
DD41,42AA) that shows a growth defect is partially defective for interaction with replication
factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase
epsilon. Two other mutants sensitive to DNA damage show no defect in vitro (Ayyagari, 1995).
To identify the regions of the proliferating cell nuclear antigen (PCNA) that are
important for function in vivo, random mutagenesis was used to isolate 10 cold-sensitive
(Cs-) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene
(POL30) in Saccharomyces cerevisiae. Unlike the Mmss mutations, the Cs- mutations
are strikingly clustered in the interdomain region of the three-dimensional PCNA
monomer structure. At the restrictive temperature, the Cs- pol30 mutants undergo a
RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in
DNA synthesis are suggested by a significant delay in the progression of synchronized
pol30 cells through S phase at the restrictive temperature. DNA repair defects are
revealed by the observation that Cs- pol30 mutants are very sensitive to the alkylating
agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive
to gamma radiation. Analysis of the chromosomal DNA in pol30 cells by
velocity sedimentation gradients shows that pol30 cells accumulate single-stranded
DNA breaks at the restrictive temperature. These results show that PCNA plays
an essential role in both DNA replication and DNA repair in vivo (Amin, 1996).
Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen
(PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is
unclear. Highly purified Pol delta from S. pombe comprises four distinct subunits of 125, 55, 54 and 22 kDa. The catalytic subunit is the Pol3 protein, while the 55, 54 and 22 kDa
subunits have been identified as Cdc1, Cdc27 (see Drosophila Cdc27) and Cdm1, respectively. Interaction studies have indicated that Pol3 interacts directly with
Cdc1, which in turn binds to Cdc27. No direct interaction between Pol3 and Cdc27 has been observed. Of the non-catalytic subunits, Cdc1 and
Cdc27 are essential proteins while Cdm1 is not. The four-subunit structure of the
S. pombe enzyme contrasts with that of Pol delta purified from mammalian cells, which is dimeric in nature, comprising homologs of Pol3
and Cdc1 only (the p125 and p48/p50 proteins), although a putative third subunit of the mammalian enzyme (p66) has recently been identified, a
homolog of the Cdc27 protein. The 54 kDa subunit of DNA polymerase delta from S. pombe
interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant
similarity to the canonical PCNA binding motif first identified in the mammalian p21Cip1 protein. This motif is both necessary and sufficient for
binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. The Pcn1 binding motif in Cdc27 is distinct from
its binding site for Cdc1 and present evidence indicates that Cdc27 can bind to Pcn1 and Cdc1
simultaneously. Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding (Reynolds, 2000).
Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits: pol 3, Cdc1, Cdc27, and Cdm1. The role of Cdc27 on the structure and activity of pol delta has been examined. The four-subunit complex is shown in this study to be monomeric in structure. The shape of Cdc27 is markedly asymmetric. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region [amino acids (aa) 1-160] binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restores processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells confers viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme (Burmudez, 2000).
An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). A 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 has been identified as a component of mouse and calf thymus pol delta. p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta can be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrate that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrate that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA (Shikata, 2001).
The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases alpha, delta, and epsilon and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min-1. This sophisticated machine has been reconstituted with purified proteins, beginning with regulated CMG assembly and activation. Replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerease epsilon catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase delta can support leading-strand synthesis, but at slower rates. DNA polymerase delta is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase epsilon engagement. It is proposed that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress (Yeeles, 2017).
Continued: mutagen-sensitive 209/PCNA Evolutionary homologs part 4/4 | back to part 1/4 | part 2/4 | Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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Proliferating cell nuclear antigen:
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