Human Proliferating Cell Nuclear Antigen Research Articles

DNA Ligase I and Proliferating Cell Nuclear Antigen Form a Functional Complex

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.

PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition.

The hHus1 and several hRad proteins are involved in the control of DNA integrity checkpoints, although the mechanisms underlying these processes are unknown. Using a yeast two-hybrid system to detect protein-protein interactions, we found that human proliferating cell nuclear antigen (PCNA), a protein known to function in both DNA replication and repair, interacts with the human checkpoint-related protein Hus1 (hHus1). In human skin fibroblast cells, exposure to ionizing radiation of hydroxyurea triggers translocation of hHus1 from the cytosol to the nucleus, where it associates with PCNA as well as another checkpoint protein, hRad9. This nuclear translocation and the complex formation or hHus1 with PCNA and hRad9 correlate closely with changes in cell cycle distribution in response to radiation exposure. These results suggest that this multi-protein complex may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

Fas antigen expression and outcome of oral squamous cell carcinoma

Paraffin sections of biopsy specimens obtained from 46 patients with oral squamous cell carcinomas were stained with both anti-peptide antibody against human Fas antigen and monoclonal mouse antibody against human proliferating cell nuclear antigen (PCNA). The patients received chemotherapy with a combination of carboplatin and peplomycin sulfate or mitomycin C and peplomycin sulfate before surgery. The relation between the expression of Fas antigen and the clinical features of each case was examined. The correlation between PCNA and Fas antigen expression was also studied. The mean PCNA labeling index of the 22 Fas-negative cases was 46.9%, which was significantly higher than that of the 24 Fas-positive cases (39.5%). Strong correlations were found between the expression of Fas antigen and the response to chemotherapy, tumor recurrence, and survival. The Fas-negative group had only a minor response to chemotherapy and a poor outcome, whereas the Fas-positive group had a better response to chemotherapy and a good outcome. Although lymph node metastasis was significantly related to survival, there was no correlation between Fas antigen expression and lymph node metastasis. The Kaplan-Meier survival curve of patients positive for Fas antigen was significantly better than that of patients negative for Fas antigen. Our results suggest that Fas antigen expression is an independent predictor of outcome whose usefulness should be evaluated in prospective studies.

The PCNA from Thermococcus fumicolans Functionally Interacts with DNA Polymerase δ

We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated Mr of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase δ (pol δ). When tested with the homologous Tfu pol on bacteriophage λ DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol δ. Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals.

Reversible Phosphorylation at the C-terminal Regulatory Domain of p21Waf1/Cip1 Modulates Proliferating Cell Nuclear Antigen Binding

The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.

Dynamics of β and Proliferating Cell Nuclear Antigen Sliding Clamps in Traversing DNA Secondary Structure

Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed.

Single nucleotide polymorphism analyses of the human proliferating cell nuclear antigen (pCNA) and flap endonuclease (FEN1) genes.

The products of proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN1) genes are multifunctional proteins that are involved in DNA replication and damage repair. Yeast models suggest association of mutant forms of PCNA and FEN1 with genomic instability. In our study, we have determined the single nucleotide polymorphisms in human PCNA and FEN1 genes. We sequenced the coding region and adjacent noncoding region of both the PCNA and FEN1 genes in 120 alleles (60 individuals). In the PCNA gene, we detected 9 sequence variants with Hardy-Weinberg allele frequency ranging from 0.008 to 0.088. No polymorphism was detected in the FEN1 gene. The sequence variants in the PCNA gene included 7 intronic single nucleotide polymorphisms (SNP) and 2 synonymous exonic SNPs. All the intronic SNPs were located in introns 1 and 4, which contain several regulatory elements involved in the control of PCNA gene expression. Six of the 7 intronic SNPs showed complete linkage disequilibrium. We confirmed this allelic linkage disequilibrium by allele-specific PCR sequencing. We genotyped 117 additional individuals belonging to 3 population subgroups using the PCR-RFLP method. Finally, to see if the detected polymorphisms are associated with any cancer type, we genotyped cases with melanomas (37 cases), breast cancers (118 cases) and lung cancers (100 cases). We did not find statistical difference in frequency of polymorphism in any cancer type compared with healthy controls, although in breast cancer the frequency was low. Our results suggest that the coding regions of the PCNA and FEN1 genes are highly conserved when compared with other DNA repair genes. The potential of some of the detected intronic polymorphisms to effect regulation of the PCNA gene expression remains to be determined.

Dual action of the adenovirus E1A 243R oncoprotein on the human proliferating cell nuclear antigen promoter: repression of transcriptional activation by p53.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants.

To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity.

In Vivo Structure of Two Divergent Promoters at the Human PCNA Locus: SYNTHESIS OF ANTISENSE RNA AND S PHASE-DEPENDENT BINDING OF E2F COMPLEXES IN INTRON 1

Proliferating cell nuclear antigen (PCNA) synthesis is strictly regulated during the cell cycle. To investigate PCNA transcriptional regulation, we have analyzed protein-DNA interactions at the promoter region and in the first intron in quiescent fibroblasts and following serum stimulation. Twenty putative protein-binding sites, distributed in two divergent promoters at the PCNA locus, were identified in vivo by genomic footprinting. These elements bind transcription factors continuously throughout the cell cycle with the exception of one E2F consensus site, located in the first intron at position +583. This E2F site becomes strongly occupied 18 h after serum stimulation, implying that an E2F activator complex plays a role in activation of the PCNA gene at the onset of S phase. We detected a 500-600-base pair-long antisense transcript by Northern blot analysis. This RNA has no apparent coding capacity and is constitutively transcribed from a promoter located within the first intron. We suggest that silencing of the PCNA gene is accomplished through base pairing between sense pre-mRNA and antisense RNA. The binding of S phase-specific E2F complexes at the +583 element may help to overcome the negative effect of the antisense transcript, which results in up-regulation of PCNA expression in proliferating cells.

Involvement of RFX1 Protein in the Regulation of the Human Proliferating Cell Nuclear Antigen Promoter

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth.

The phosphoprotein NS5A of hepatitis C virus has recently been suggested to control PKR protein kinase for resistance to interferon. To investigate other functions of NS5A, studies were initiated on the regulation of transcription of important cellular genes and of cell growth by this protein. The results suggested that NS5A protein represses transcription of the cell cycle regulatory gene p21WAF1, while it activates the human proliferating cell nuclear antigen gene in murine fibroblasts and human hepatoma cells. Furthermore, introduction of NS5A into murine fibroblasts (NIH3T3) promoted anchorage-independent growth and tumour formation in nude mice. Thus, NS5A appears to exhibit a role in cell growth regulation.

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764–5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template–primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as α, β, γ, δ, ε and ζ and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3′–5′ exonuclease activities as shown by the chromatographies.

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen.

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. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered 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.

Differential Regulation of p53-dependent and -independent Proliferating Cell Nuclear Antigen Gene Transcription by 12 S E1A Oncoprotein Requires CBP

The tumor suppressor protein p53 and the adenoviral 12 S E1A oncoprotein are both known to elicit their biological effects mainly by regulating the transcription of important cellular genes. The human proliferating cell nuclear antigen (PCNA) gene is a transcriptional target of both p53 and E1A. We have analyzed the effects of p53 and 12 S E1A, separately as well as together, on PCNA gene transcription. Our results showed that whereas both p53 and 12 S E1A separately activated PCNA transcription, 12 S E1A repressed p53-mediated transcriptional activation. Thus, 12 S E1A uses a dual strategy of transcriptional activation and repression to take control of the cellular PCNA gene regulation. The cyclic AMP-response element in the PCNA core promoter, besides being crucial for basal transcription, synergizes with p53 to activate transcription. The cyclic AMP response element-binding protein (CREB)-binding protein (CBP) is an essential component of both the transcriptional activation and repression by E1A. Our data demonstrate for the first time that E1A can modulate CBP function to activate PCNA transcription, while at the same time repressing p53-mediated activation by disrupting CBP interaction with p53, thereby uncoupling PCNA transcription from the regulatory effects of p53.

'Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain.

Tumor-derived p53 mutants can transcriptionally activate a number of promoters of genes involved in cellular proliferation. For this transactivation, mutant p53 does not use the wild-type p53 DNA-binding site, suggesting a mechanism of transactivation that is independent of direct DNA binding. Here we describe our analysis of the domain requirements for mutant p53 to transactivate promoters of the human epidermal growth factor receptor (EGFR), human multiple drug resistance 1 (MDR-1) and human proliferating cell nuclear antigen (PCNA) genes. We also report the identification of a structural domain required for the 'gain of function' property of mutant p53-281G. 'Gain of function' is measured as the tumorigenicity (in nude mice) of 10(3) murine cells expressing mutant p53 constitutively. We have generated internal deletion mutants of p53-281G deleting conserved domains I, II, III, IV and V, individually. We have also generated one deletion mutant eliminating amino acids 100 through 300 that removes four of the five conserved domains (II - V); another mutant, p53-281G del 393-327, deletes the oligomerization and nonsequence-specific nucleic acid-binding domains of p53. For the EGFR and MDR-1 promoters, all these mutants have significantly lower transactivation ability than intact p53-281G. These deletion mutants, however, significantly activated the pCNA promoter, suggesting that the mechanism of transactivation of the PCNA promoter is different from that of the EGFR and MDR-1 promoters. When expressed constitutively in 10(3) cells, p53-281G del 393-327 was found to be defective in inducing tumor formation in nude mice although intact p53-281G was very efficient. Thus, our results suggest that structural domains near the C-terminus are needed for 'gain of function'.

Staurosporine-sensitive protein phosphorylation is required for postreplication DNA repair in human cells

DNA repair is an important factor of stability of pro- and eukaryotic genomes which plays a central role in mutagenesis and carcinogenesis. Genetic control of nucleotide excision repair (NER) in mammalian cells is well studied, but little is known about molecular mechanisms of postreplication repair (PRR) which allows bypass of base lesions in template strands after DNA replication. In Saccharomyces cerevisiae PRR is controlled by the RAD6/RAD18 pathway which involves POL30 gene encoding proliferating cell nuclear antigen (PCNA), and in human cells PCNA is known to be closely associated with the newly replicated chromatin where PRR probably takes place. In UV-irradiated human cells distinct PCNA foci may be detected in some cells which accumulate phosphorylated breast cancer susceptibility protein BRCA1 and another protein BARD1. Human PCNA is also known to be phosphorylated after UV-irradiation. In this study we found that the known inhibitor of protein kinases staurosporine supresses PRR in NER-deficient cells which is consistent with the view that BRCA1 and PCNA are required for PRR. We also have shown that the distinct PCNA foci in UV-irradiated NER-deficient cells are actually associated with the newly replicated chromatin. Since RAD18 protein is not essential for normal DNA replication and directly controls PRR in yeast, we analysed whether this protein as well as its human homologs (HR18A and HR18B) have common domains with BRCA1 and BARD1. It is found that HR18A has a subregion of homology to BARD1 and HR18A-to BRCA1. Taken together the results indicate that BRCA1 and BARD1 may be involved in PRR in human cells.

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.

Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107.

The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425-437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107's ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107's ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732-3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase δ or ε to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.