Proliferating cell nuclear antigen
Following genomic damage, the cessation of DNA replication is co-ordinated with
onset of DNA repair; this co-ordination is essential to avoid mutation and genomic
instability. To investigate these phenomena, proteins that interact
with PCNA have been analyzed. One such protein
is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while
allowing repair to continue. An interaction has been identified between PCNA and the
structure specific nuclease, Fen1, which is involved in DNA replication. Deletion
analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within
Fen1 and its homologs a small region (10 amino acids) is sufficient for PCNA
binding; this region contains an 8 amino acid conserved PCNA-binding motif. This motif
shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding
peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the
Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA
synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding
to the same site on PCNA may provide a mechanism to co-ordinate the functions of
PCNA in DNA replication and repair (Warbrick, 1997).
Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that
is required for DNA replication during S phase of the cell cycle and for resynthesis
during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease
1 (FEN-1), a structure-specific endonuclease involved in DNA replication. There is a direct physical interaction of PCNA with xeroderma pigmentosum (XP) G,
a structure-specific repair endonuclease that is homologous to FEN-1. A 28-amino acid region of human FEN-1 (residues 328-355) and a 29-amino
acid region of human XPG (residues 981-1009) contain the PCNA binding
activities. These regions share key hydrophobic residues with the PCNA-binding
domain of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1); all three
compete with one another for binding to PCNA. A conserved arginine in FEN-1
(Arg339) and XPG (Arg992) was found to be crucial for PCNA binding activity.
R992A and R992E mutant forms of XPG fail to fully reconstitute nucleotide
excision repair in an in vivo complementation assay. These results raise the possibility
of a mechanistic linkage between excision and repair synthesis mediated by
PCNA (Gary, 1997).
A two-hybrid system was used to screen yeast and human expression libraries for
proteins that interact with mismatch repair proteins. PCNA was recovered from both
libraries and shown in the case of yeast to interact with both MLH1 and MSH2. MLH1 (mutL homolog) is a protein with sequence similarity to DNA mismatch
repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). MSH2 is a homolog of the bacterial DNA mismatch protein MutS. A
yeast strain containing a mutation in the PCNA gene has a strongly elevated mutation
rate in a dinucleotide repeat; the rate is not further elevated in a strain also
containing a mutation in MLH1. Mismatch repair activity was examined in human cell
extracts using an assay that does not require DNA repair synthesis.
Activity is
inhibited by p21WAF1 or by a p21 peptide,
both of which bind to PCNA; activity is
restored to inhibited reactions by addition of PCNA. The data suggest a PCNA
requirement in mismatch repair at a step preceding DNA resynthesis. The ability of
PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and
replication and may be relevant to the roles of mismatch repair proteins in other DNA
transactions (Umar, 1996).
Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in
lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational
information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual
DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage
junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA)
and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA
repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat
expansions as seen in some cancers and genetic diseases (Hosfield, 1998).
Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results are reported. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular ß sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites (Chapados, 2004).
Flap EndoNuclease-1 (FEN-1) is a structure-specific nuclease that is central to both DNA replication and repair processes. During DNA replication and repair, a complex that includes both FEN-1 and the 'sliding clamp' accessory protein PCNA removes RNA primers or damaged DNA, generating a product for ligation by DNA ligase I. Several lines of evidence underscore the importance of FEN-1 activity in DNA replication and repair pathways. FEN-1 homozygous knockouts are lethal in mice, and mice heterozygous for functional FEN-1 (FEN-1/null) exhibit accelerated tumor growth. Deletions of FEN-1 in Saccharomyces cerevisiae (rad27) cause replication and repair defects, including increased sensitivity to UV light and chemical mutagens, genomic instability, increased tri-nucleotide repeat expansion, and destabilization of telomeric repeats. These data highlight the importance of FEN-1 function in cells and make FEN-1 a potential cancer susceptibility gene (Chapados, 2004 and references therein).
The FEN-1 class of structure-specific 5' nucleases occurs in all domains of life. Unlike endonucleases that recognize a specific DNA sequence, FEN-1 recognizes a specific DNA structure, independent of the DNA sequence. Specifically, FEN-1 and related 5' nucleases recognize a branched DNA structure consisting of a single unpaired 3' nucleotide (3' flap) overlapping with a variable length region of 5' single-stranded DNA (5' flap). This 'double-flap' or 'overlap-flap' structure results from DNA polymerase activity that displaces damaged DNA or RNA creating a ssDNA 5' flap. The newly synthesized DNA and the displaced region compete for base pairing with the template strand, resulting in the formation of the double-flap structure. FEN-1 cleaves this substrate after the first base pair preceding the 5' flap to remove the ssDNA 5' flap and create a nicked DNA product (Chapados, 2004 and references therein).
The dramatic increase in FEN-1 cleavage specificity for the double-flap structure suggests that FEN-1 specifically binds to the 3' flap. Although several structures of FEN-1 homologs exist, a specific binding site for the 3' flap has not been identified. Biochemical results also suggest that conformational changes in flexible loop regions are required for catalysis. However, in the absence of structural information on FEN-1 and DNA conformational changes, efforts to model FEN-1 interactions with DNA cannot account for the structure-specific activity observed in biochemical assays (Chapados, 2004 and references therein).
In cells, FEN-1 forms a complex with PCNA, which exists as a ring-shaped homotrimer in solution. PCNA is loaded onto DNA in an ATP-dependent reaction catalyzed by replication factor C (RF-C). After loading, PCNA encircles dsDNA and can slide freely along it. In this way, PCNA can act as a molecular adaptor or 'sliding clamp' that localizes bound proteins to DNA (Chapados, 2004 and references therein).
FEN-1 binds to PCNA through a conserved consensus motif. Crystal structures of peptides derived from p21waf1/cip1 and other proteins bound to PCNA show that half of the consensus PCNA binding motif adopts a helical conformation, placing the conserved hydrophobic residues (LXXFF) on the same face of the helix. This structure facilitates interactions with a hydrophobic pocket on the PCNA surface formed by residues in the interdomain connecting loop of PCNA. The conservation of the PCNA binding consensus motif suggests a generic way in which several different enzymes can bind to PCNA (Chapados, 2004 and references therein).
PCNA stimulates FEN-1 activity by up to 50-fold in vitro. Even when all residues of the PCNA binding motif are mutated to alanine, preventing the known hydrophobic interaction, PCNA still stimulates FEN-1 activity. Furthermore, when PCNA is loaded onto DNA by RF-C, the C terminus of PCNA mediates interactions with FEN-1. These results suggest that interactions with the C terminus of PCNA either involve FEN-1 residues located outside the currently defined PCNA binding motif or are somehow independent of the amino acid sequence (Chapados, 2004 and references therein).
Current knowledge of FEN-1 interactions with DNA and PCNA therefore raises three critical questions: (1) How does FEN-1 recognize the DNA 3' flap? (2) How does recognition of the 3' flap aid structure-specific catalysis? (3) How does FEN-1 interaction with PCNA increase FEN-1 activity? These key questions are addressed by providing cocrystal structures of Archaeoglobus fulgidus FEN-1 bound to DNA and of two FEN-1 peptides bound to A. fulgidus PCNA, coupled with fluorescence energy transfer (FRET) activity, and mutational analyses (Chapados, 2004).
It is proposed that FEN-1 binding to the 3' flap, in addition to hydrophobic contacts with the exposed bases, anchors the DNA in a defined orientation. The structural and FRET results suggest that the DNA is kinked, such that when the 3' flap is bound, the scissile phosphate is positioned near the active site. Because of the 25 Å separation between the 3' flap binding site and the active site, FEN-1 could track along the 5' flap, but not efficiently catalyze phosphodiester cleavage until 3' flap binding promotes ordering of the helical clamp over the properly positioned substrate. The results presented here argue that 3' flap binding defines a registration point that may provide a 'molecular ruler' to ensure that the scissile phosphate is positioned near the active site. Binding of the 3' flap could then promote closing of an ordered, helical clamp over the active site. This DNA-dependent, conformational change would exclude bulk solvent from the active site and facilitate precise cleavage of the substrate at the base preceding the flap junction (Chapados, 2004).
Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free
SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding
the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen.
Using deletion mutants of RF-Cp145, the DNA binding domain of
RF-Cp145 has been localized to amino acid residues 369-480. This domain is conserved among
prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent
in other subunits of RF-C. The PCNA binding domain maps to amino acid residues
481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of
RF-Cp145 inhibits at least three functions of RF-C: (1) in vitro DNA replication of
SV40 origin-containing DNA; (2) RF-C-dependent loading of PCNA onto DNA, and
(3) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145
localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In
contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in
vitro or in vivo. It is concluded that amino acid residues 481-728 of human
RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in
DNA replication in vivo (Fotedar, 1996).
Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or
epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C
(RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. The identification of two separate peptide regions of human PCNA spanning amino
acids 36-55 and 196-215 that bind RFC was carried out by using the surface plasmon resonance technique. Site-directed
mutagenesis of residues within these regions in human PCNA identifies two specific sites that affected the
biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine
significantly impairs the ability of PCNA to (1) support the RFC/PCNA-dependent polymerase
delta-catalyzed elongation of a singly primed DNA template; (2) stimulate RFC-catalyzed DNA-dependent
hydrolysis of ATP; (3) be loaded onto DNA by RFC, and (4) activate RFC-independent polymerase
delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also
reduces the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a
singly primed DNA template. However, this mutation does not significantly alter the ability of PCNA to
stimulate DNA polymerase delta in the absence of RFC but substantially lowers the efficiency of
RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of
PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex
in a manner essential for processive DNA synthesis (Zhang, 1999).
The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 is still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreases both modes of interaction with PCNA and results in replication and repair defects in vivo (Gomes, 2000).
Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, a variety of flap substrates were tested. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, these results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency (Tom, 2000).
Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Four amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X3(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although the results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, an in vitro assay showed that hPCNA fails to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities are significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retain PCNA-driven nuclease activity stimulation. It is therefore concluded that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region (Frank, 2001).
Genome instability is a hallmark of cancer and plays a critical role in generating the myriad of phenotypes selected for during tumor progression. However, the mechanisms that prevent genome rearrangements remain poorly understood. To elucidate the mechanisms that ensure genome stability, a collection of candidate genes was screened for suppressors of gross chromosomal rearrangements (GCRs) in budding yeast. One potent suppressor gene encodes Elg1, a conserved but uncharacterized homolog of the large Replication factor C subunit Rfc1 and the alternative RFC subunits Ctf18/Chl12 and Rad24. Replication factor C (RFC) is a complex of distinct subunits that functions as a clamp loader, facilitating the loading of proliferating cell nuclear antigen (PCNA) onto DNA. The results are consistent with the hypothesis that Elg1 forms a novel and distinct RFC-like complex in both yeast and human cells. Elg1 is required for efficient S phase progression and telomere homeostasis in yeast. Elg1 interacts physically with the PCNA homolog Pol30 and the FEN-1 homolog Rad27. The physical and genetic interactions suggest a role for Elg1 in Okazaki fragment maturation. Furthermore, Elg1 acts in concert with the alternative Rfc1-like proteins Rad24 and Ctf18 to enable Rad53 checkpoint kinase activation in response to replication stress. Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex. Furthermore, it is proposed that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation (Kanellis, 2003).
Gadd45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain
other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to
cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), Gadd45 has been
associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of
Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulates DNA
excision repair in vitro and inhibits entry of cells into S phase. These results establish Gadd45 as a
link between the p53-dependent cell cycle checkpoint and DNA repair (Smith, 1994).
Antibodies raised against the growth arrest and
DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a
protein involved in DNA replication and repair. Gadd45 can directly bind
to PCNA. In a far Western assay, a Gadd45 bacterial expression
vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein
was used to detect filter bound PCNA protein, while filter bound Gadd45 protein can also be
detected by free PCNA molecules. Gadd45 competes with p21 for binding to
PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. p21 appears
to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption is also
observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A
(XP-A) cells (I. Chen, 1995b).
Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1; Drosophila homolog - Double parked) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication. Replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1-Cul4(DDB1) ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator (Arias, 2006).
Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. The N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage (Senga, 2006).
At low levels, wild-type p53
transactivates the human proliferating cell nuclear antigen (PCNA) promoter. When expressed at a
similar level, the tumor-derived p53 mutants do not transactivate the PCNA promoter. A p53-binding site is present on the human PCNA promoter; here, p53 interacts sequence
specifically. The binding site functions as a
wild-type p53 response element in either orientation. Deletion of the p53-binding site renders the
PCNA promoter p53 nonresponsive, showing that wild-type p53 transactivates the PCNA
promoter by binding to the site. At a higher concentration, wild-type p53 inhibits the PCNA
promoter but p53 mutants activate the promoter. Transactivation by p53 mutants does not require the p53-binding
site. These observations suggest that moderate elevation of the cellular wild-type p53 level induces
PCNA production to help in DNA repair (Shivakumar, 1995).
Examination of the human PCNA promoter DNA sequence reveals a site with
homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site
intact bind p53 in vitro and are transcriptionally activated by wild-type p53 in transient
expression assays in SAOS-2 cells. The resident p53-binding site can be functionally substituted
by a p53-binding site from the ribosomal gene cluster. A plasmid expressing a
mutated version of p53 derived from a patient with Li-Fraumeni syndrome fails to activate the
PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA
promoter by the p53-binding sequence correlates with the status of p53. Activation of the PCNA
promoter by wild-type p53 depends upon the level of p53 expression. This concentration
dependence and cell type specificity reconciles the observations presented here with prior results
indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism
whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage (Morris, 1996).
The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a
potent transactivator of both viral and cellular gene expression. The ability of Tax to
transform cells is believed to depend on its transactivation of
cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen
(PCNA) is intimately linked to cell growth and DNA replication and repair. By testing
a series of PCNA promoter deletion constructs, it was demonstrated that the
PCNA promoter can be transactivated by Tax. The smallest construct that is
activated does not include the ATF/CRE binding site at nucleotide -50, and mutations in
the ATF/CRE element in the context of a larger promoter are still activated by Tax.
In addition, a Tax mutant that is defective for activation of the CRE pathway retains
the ability to activate the -397 promoter construct. When a series of linker scanner
mutations that span the region from nucleotide -45 to -7 are assayed, mutations in
and around a repeat sequence are found to abolish Tax transactivation. Multimerized
copies of either half of the repeat are Tax responsive. A single protein complex is
shown to bind specifically to the Tax-responsive region; the binding of this
complex is enhanced in the presence of Tax. These results demonstrate that the
PCNA promoter contains a Tax-responsive element located between nucleotides -45
and -7, whose sequence is different from those of other, previously identified
Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play
an important role in cellular transformation by HTLV-1 (Ressler, 1997).
Interleukin 2 (IL-2) stimulates T-lymphocyte proliferation and induces the expression
of proliferating cell nuclear antigen (PCNA). Previously, deletion analysis suggested that cis-element(s) in the
proximal region of the PCNA promoter (-40 to +143) are required for IL-2 induction
in cloned T lymphocytes. The sequence 5'-TTGCGGGC-3' located at +10 to +17 is
similar to the E2F consensus binding site and is required for optimal PCNA promoter
activity. In IL-2-stimulated T cells, nuclear proteins are induced to bind to this
sequence as demonstrated using electrophoretic mobility shift assay (EMSA),
competition EMSA, and methylation interference analysis. A 180-kDa polypeptide binds specifically to the PCNA E2F-like
sequence. The protein bound to the PCNA E2F-like site is not
one of the transcription factor E2F proteins. These results demonstrate that the E2F-like
sequence and the protein(s) binding to it are required for optimal PCNA promoter
activity and IL-2 induction of PCNA expression (Huang, 1996).
Binding of the C-terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4-E1A fusion
protein abolishes conserved region (CR) 1-dependent transcription activation. In contrast, a
non-promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the
proliferating cell nuclear antigen (PCNA) promoter. CtBP is shown here to bind the histone
deacetylase HDAC1, suggesting that a promoter targeted CtBP-HDAC1 complex can silence
transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP
binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the
CtBP-HDAC1 complex from the promoter (Sundqvist, 1998).
Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. The mating phenotypes of haploid cell types in Saccharomyces require transcriptional repression of mating-type genes at HML and HMR. These loci contain mating-type genes identical to those at the expressed MAT locus, but are silenced in a mechanism comparable to
position effect variegation in larger eukaryotes. The silenced loci are flanked by sequence elements termed silencers that contain
binding sites for the proteins Rap1 and Abf1 as well as for the origin recognition complex (ORC). The silencer binding proteins likely serve to attract other silencing
factors, the silent information regulator (Sir) proteins, to form a specialized chromatin structure in the repressed regions that renders them inaccessible to transcription,
thus achieving silencing. This study examined the effects on HMR locus silencing of 41 mutations in genes with roles in replication, the cell cycle, and DNA repair. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicate that the mutant HMR locus is replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations requires the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed. One common feature of most of the suppressors is that the respective proteins function
close to the moving replication fork. The suppression by some but not other mutations in proteins at the replication fork may suggest that these proteins are in some
way specifically involved in the formation of silenced chromatin, perhaps by communicating to other protein complexes that establish silenced chromatin after the
passage of replication forks. Because loss of function mutations in these genes led to silencing in a subset of cells in the population, one would infer that in wild-type
cells, these proteins promote the assembly of newly replicated DNA into euchromatin (Ehrenhofer-Murray, 1999).
Chromatin assembly factor 1 (CAF-1- see Drosophila Chromatin assembly factor 1 subunit) is required for inheritance of epigenetically determined chromosomal states in vivo
and promotes assembly of chromatin during DNA replication in vitro. After DNA replication,
the replicated (but not unreplicated) DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell
nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for
chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the
replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of
DNA replication in cells. It is suggested that PCNA and CAF-1 connect DNA replication to chromatin assembly and the
inheritance of epigenetic chromosome states (Shibahara, 1999).
CAF-1 assembles nucleosomes during DNA replication in vitro on both the leading and lagging strands
behind a replication fork. Nucleosome assembly
is an ordered process, with histones H3 and H4 first loaded onto the DNA during replication in a
CAF-1-dependent manner, and soon thereafter, histones H2A and H2B are added to form a mature
nucleosome. This is most likely how the bulk of
de novo nucleosome assembly occurs during S phase.
In the current paper, however, it has been
demonstrated that nucleosome assembly can be temporally dissociated from passage of the DNA
replication fork. Addition of CAF-1 after completion of DNA replication allows nucleosome assembly
on the replicated DNA, but not on the unreplicated DNA that is present in the same reaction. Thus,
the replicated DNA is marked or imprinted for subsequent CAF-1-dependent processes. It has been
suggested that a component of the DNA replication fork mediates CAF-1 function, and it now seems likely that
PCNA is that factor (Shibahara, 1999 and references).
The PCNA clamp is involved in the synthesis of both the leading and lagging strands at the DNA
replication fork.
On the leading strand, PCNA
associates with DNA polymerase and promotes continuous synthesis of long DNA strands in a
processive manner. Thus, one PCNA clamp is needed per initiation event. In contrast to this, during lagging-strand synthesis, DNA replication occurs by the discontinuous production of
Okazaki fragments, a process that involves a PCNA-dependent polymerase switching mechanism. In this case, one clamp needs to be
loaded for every Okazaki fragment or approximately every 100-200 bp. On both strands, PCNA
is loaded by RFC at a primer-template DNA junction that is later recognized by the DNA polymerase.
When the polymerase completes DNA synthesis, it dissociates from the DNA and leaves
the ring-shaped clamp topologically linked to the duplex, replicated DNA. This implies that the rate-limiting step
for removal of PCNA from the replicated DNA (and hence loss of the replication 'imprint') is the
ATP-dependent unloading of PCNA by RFC. Thus, RFC has the potential to regulate the duration of
the PCNA marking on replicated DNA. This might occur in a locus-specific manner in
the genome, providing a mechanism for gene-specific modulation of chromatin structure (Shibahara, 1999).
It is suggested that CAF-1-mediated nucleosome assembly normally occurs on both the leading and
lagging strands immediately after passage of the DNA replication fork, as occurs in vitro when CAF-1
is present during DNA replication. Indeed, CAF-1 localizes to the sites of DNA replication in cells. But the data described here suggest that an alternative, postreplicative
mechanism may also operate that could provide opportunities for asymmetric inheritance of chromatin
states. If the PCNA that is used for Okazaki fragment synthesis were to remain associated with the
lagging-strand product for some time after DNA replication, the amount of PCNA bound to the two
sister chromatids would be inherently asymmetric. Since CAF-1 binding to PCNA can allow
chromatin assembly after DNA replication, this situation would offer considerable opportunities for the
establishment of an asymmetric chromatin structure on the two sister chromatids prior to the division of
proliferating stem cells. Such chromatin complexes, if transferred into the daughter cells, could provide the
foundation for phenotypic asymmetry of sister cells during development (Shibahara, 1999).
Although chromatin states must normally be inherited by both daughter cells, it has long been suspected
that DNA replication provides a window of opportunity for changes in chromatin structures that might
affect gene expression.
Indeed, replication-coupled chromatin assembly suppresses basal transcription. Recent studies in the budding yeast Saccharomyces cerevisiae have
showen that CAF-1 is required for the stable inheritance of transcriptionally repressed chromatin
structures at telomeres and HM loci. In the absence of CAF-1, the
expression of genes near the telomeres is variegated in a population of cells. It is suggested that the
recruitment of CAF-1 by PCNA is required for suppression of this form of position effect variegation.
Support for a role for PCNA in suppression of position effect variegation comes from genetic studies in
Drosophila. A mutant in Drosophila PCNA, mus209, is a suppressor of position effect variegation and exhibits DNA repair defects that overlap with the repair defects
seen in yeast lacking CAF-1. The mus209 mutant also causes strong
enhancement of homeotic transformation in a trans-heterozygous crm;mus209 mutant
(Yamamoto, 1997 ). The cramped (crm) gene is a Polycomb-group (Pc-G) gene, and
some crm mutants show suppression of position effect variegation. PCNA and the CRM protein
colocalize in nuclei of proliferating cells. These phenotypes may reflect the role of PCNA in promoting
efficient coupling between DNA replication and chromatin assembly (Shibahara, 1999 and references).
The inheritance of epigenetically determined states in budding and fission yeasts may also occur by
similar mechanisms. In the fission yeast S. pombe, replication proteins such as DNA polymerase,
chromatin proteins Swi6 and Clr4, and histone deacetylases are involved in the epigenetic inheritance of
the mating type silent chromatin and centromeric heterochromatin. It is
possible that PCNA and CAF-1 mediate these effects. In the budding yeast S. cerevisiae, position
effect variegation occurs at telomeres, and
the establishment of chromatin repression at the mating type loci requires passage through S phase. Furthermore, heterochromatin at the mating type loci (HM) is uniquely
acetylated at lysine-12 of histone H4, and a variety of mutations in the
acetylated lysines of histones H3 and H4 result in defects in silencing at HM loci and telomeric reporter
genes. Since CAF-1 associates with specific forms of acetylated histones, the PCNA-CAF-1-linked chromatin assembly may be involved in
assembly of heterochromatin and epigenetically determined chromosomal states.
PCNA interacts with the DNA-(cytosine-5)-methyltransferase at sites of DNA replication in
mammalian cells, and this interaction likely mediates the inheritance of DNA methylation patterns. It is suggested that the PCNA molecules that remain associated with the
replicated DNA might provide a common platform for both chromatin assembly and for DNA
methylation. In some situations, this could allow preferential methylation of one sister chromatid over
the other, as has been proposed for chromatin assembly (Shibahara, 1999 and references).
Formation of a heterochromatin-like structure results in transcriptional silencing at the HM mating-type loci and telomeres in Saccharomyces cerevisiae. Once
formed, such epigenetically determined structures are inherited for many mitotic divisions. PCNA is important in many aspects of DNA metabolism, including DNA replication and DNA repair in eukaryotes. Human PCNA interacts with CAF-1, a
three-subunit protein, to couple DNA replication or DNA repair to nucleosome deposition. In yeast, deletion of any genes encoding CAF-1 subunits (CAC1, 2
and 3) results in partial loss of silencing at telomeres and silent mating-type loci. Furthermore, mutations in the Drosophila PCNA gene mus209 suppress position
effect variegation. Therefore tests were performed to see whether mutations in yeast S. cerevisiae PCNA affect silencing.
Mutations in the proliferating cell nuclear antigen
(PCNA), an essential component at the DNA replication fork, reduce repression of genes near a telomere and at the silent mating-type locus, HMR. The pol30-8
mutant displays coexistence of both repressed and de-repressed cells within a single colony when assayed with the ADE2 gene inserted at HMR.
Unlike pol30-8, the pol30-6 and pol30-79 mutants partially reduce gene silencing at telomeres and the HMR and synergistically decreased silencing in cells lacking
chromatin assembly factor 1 (CAF-1). All silencing defective mutants show reduced binding to CAF-1 in vitro and altered chromatin association of the CAF-1
large subunit in vivo. Thus two classes of pol30Delta mutants have been discovered, on the basis of their combined effect with cac1 on TPE and HMR silencing, their effect on HMR silencing,
and their location on the three-dimensional structure of PCNA. Genetic analysis suggests that PCNA is involved in silencing
CAF-1, both dependently and independently, providing direct mechanisms to link inheritance of DNA to the propagation of epigenetic states of gene expression in eukaryotes, a process believed to
determine cell fate in complex organisms. Thus, PCNA participates in inheritance of both DNA and epigenetic chromatin structures during the S phase of the cell cycle, the latter by at least two mechanisms (Zhang, 2000).
Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p (see Drosophila Asf1) and chromatin assembly factor I (CAF-I, a heterotrimeric protein complex encoded by the CAC1-3 genes). CAF-I performs the first step of nucleosome formation, deposition of the histone (H3/H4)2 tetramer onto DNA. CAF-I delivers histones to DNA molecules that have been recently replicated either bidirectionally or during DNA repair; it is targeted to replicated DNA via a direct interaction with proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor. Both cacDelta asf1Delta and cacDelta hirDelta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. Biochemical and genetic studies of yeast Asf1p have revealed links to Hir protein function. In vitro, an active histone deposition complex is formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generates micrococcal nuclease-resistant DNA in the absence of DNA replication and stimulates nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p binds to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contribute to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminates telomeric gene silencing in combination with pol30-8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevent Asf1/Hir proteins from contributing to silencing. It is concluded that yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity (Sharp, 2001).
Continued: mutagen-sensitive 209/PCNA Evolutionary homologs part 3/4 | part 4/4 | back to part 1/4 | Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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