Human Proliferating Cell Nuclear Antigen Research Articles

The Interdomain Connector Loop of Human PCNA Is Involved in a Direct Interaction with Human Polymerase δ

Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase delta (pol delta) and polymerase epsilon. We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol delta, maps to residues 121-135, which overlap the interdomain connector loop of PCNA (residues 119-133). We have mutagenized residues 122-133 of human PCNA. The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity. The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol delta in several different assays. These mutations affected the interactions between PCNA and pol delta as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays. The findings show that the interdomain connector loop region is involved in binding of pol delta. This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol delta.

Transcriptional coactivator cAMP response element binding protein mediates induction of the human proliferating cell nuclear antigen promoter by the adenovirus E1A oncoprotein.

The proliferating cell nuclear antigen (PCNA), a crucial component of eukaryotic cell cycle and DNA replication complexes, is induced by the adenovirus E1A 243R oncoprotein through a cis-acting element termed the PERE (PCNA-E1A responsive element). The PERE contains a sequence homologous to an activating transcription factor (ATF) motif, and ATF-1 is a major component of PERE-protein complexes. We have identified a second PERE-binding protein, the cAMP response element binding protein (CREB) transcription factor, which forms heterodimers with ATF-1 at this site. CREB, but not ATF-1, is able to mediate transactivation of a minimal PCNA-chloramphenicol acetyltransferase reporter by E1A 243R. Further analysis revealed that the transcriptional coactivator, the CREB-binding protein (CBP), associates with PERE-related complexes, and that CBP is able to mediate a strong transactivation response to E1A 243R at the PCNA promoter. Experiments conducted with mutants in the E1A or CREB components support a model whereby E1A 243R transactivates the PCNA promoter via a CBP-CREB-PERE pathway. These findings delineate a paradigm by which E1A 243R can target and transactivate specific DNA promoter sequences.

Replication Factor C Interacts with the C-terminal Side of Proliferating Cell Nuclear Antigen

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.

Cloning, sequencing, and chromosomal localization of two tandemly arranged human pseudogenes for the proliferating cell nuclear antigen (PCNA).

We have characterized a human genomic clone carrying two pseudogenes for the proliferating cell nuclear antigen (PCNA), which were tandemly arranged on human Chromosome (Chr) 4. One is a processed pseudogene that showed a 73% nucleotide homology to the human PCNA cDNA and possessed none of the introns existing in the functional PCNA gene. This pseudogene presumably arose by reverse transcription of a PCNA mRNA followed by integration of the cDNA into the genome. The other is a 5' and 3' truncated pseudogene that showed a nucleotide homology to a 3' region of the exon 4 and to a 5' region of the exon 5 of the PCNA gene and did not have the intronic sequence between the exons 4 and 5. Both pseudogenes had the same nucleotide deletion as compared with the human functional PCNA gene. A phylogenetic analysis of PCNA gene family, including the functional PCNA gene and another PCNA pseudogene located on a different chromosome, revealed that the truncated pseudogene exhibits the closest evolutionary relationship with the processed pseudogene, suggesting that the truncated pseudogene was generated by duplication of the processed pseudogene after translocation to Chr 4. Furthermore, fluorescence in situ hybridization revealed that these pseudogenes are located on the long arm of Chr 4, 4q24.

P21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Structure of the C-Terminal Region of p21WAF1/CIP1 Complexed with Human PCNA

The crystal structure of the human DNA polymerase δ processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21WAF1/CIP1 has been determined at 2.6 Å resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a β sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21–PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.

PCNA mRNA Has a 3′ UTR Antisense to Yellow Crescent RNA and Is Localized in Ascidian Eggs and Embryos

The myoplasm is a localized cytoplasmic region that is involved in axis determination, gastrulation, muscle cell specification, and the pattern of cell divisions during ascidian development. The noncoding yellow crescent (YC) RNA is localized in the myoplasm, but the function of this transcript is unknown. Probes containing the 3′ region of YC RNA hybridize to other RNAs in ascidian eggs. A cDNA library from the ascidianStyela clavawas screened with a YC probe to identify maternal YC-related RNAs. This screen resulted in isolation of ScYC26b, a cDNA clone encoding the ascidian proliferating cell nuclear antigen (PCNA). The PCNA mRNA has a long 3′ untranslated region containing a 521-nucleotide sequence with antisense complementarity to part of the 3′ region of YC RNA. The PCNA and YC genes appear to be single copy and may overlap in their 3′ regions on opposite DNA strands. The ascidian PCNA protein has 61, 69, and 71% amino acid identity to theDrosophila, Xenopus,and human PCNAs, respectively.S. clavaembryos contain maternal and zygotic PCNA mRNAs. Maternal PCNA mRNA is localized in the ectoplasm, a cytoplasmic region that is segregated to cell lineages that proliferate extensively during embryogenesis, and is depleted in the myoplasm, which is segregated to cell lineages that undergo fewer divisions. Zygotic PCNA mRNA is confined to the developing nervous system and is still abundant after the neural cells have ceased to proliferate. PCNA protein, detected with PC10 monoclonal antibody, is also excluded from the myoplasm. These results show that the 3′ UTR of PCNA mRNA is antisense and complementary to YC RNA and suggest that differential cell proliferation in the embryo may be limited by localization of maternal PCNA mRNA and protein. Furthermore, zygotic PCNA may have a novel role in neural development in the tadpole larva.

Determination of the Epitope of an Inhibitory Antibody to Proliferating Cell Nuclear Antigen

Proliferating cell nuclear antigen (PCNA) is an essential component for the normal processive DNA synthesis by DNA polymerase δ and is also required for DNA excision repair. Human PCNA autoantisera has been shown to inhibit the function of PCNAin vitro,in contrast to induced antibodies. A monoclonal IgG2PCNA antibody, 74B1, that effectively inhibited DNA replicationin vitrowas identified. The inhibitory effect was dose dependent and using synthetic overlapping peptides of PCNA the 74B1 epitope was mapped to aa 121–135, a region of the PCNA protein containing the interdomain connector implicated in intermolecular interactions. Interestingly, a neighboring and partly overlapping peptide, aa 111–125, contains an immunodominant region recognized by a number of monoclonal PCNA antibodies with no inhibitory effect on DNA synthesis. The 74B1 antibody is a potentially useful antibody in future studies of PCNA and its interaction in complexes associated with cell cycle progress, DNA replication, and DNA repair.

Two Pathways for Base Excision Repair in Mammalian Cells

Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Implications for antisense therapy of restenosis after angioplasty.

Antisense oligonucleotides have been used in animals to inhibit the accumulation of vascular smooth muscle cells (VSMCs) after arterial injury. This has raised prospects for an oligonucleotide-mediated approach to prevent restenosis in patients undergoing angioplasty. However, little is known about the processing of oligonucleotides by human VSMCs or their bioavailability in human atherosclerotic tissue. Oligonucleotides were synthesized with a mixture of unmodified and sulfur-modified linkages (S-chimeric oligonucleotides). These were more stable than unmodified oligonucleotides and could be recovered from within human VSMCs after 36 hours. Oligonucleotide antisense to human proliferating cell nuclear antigen mRNA specifically reduced DNA synthesis (P < .01) and proliferating cell nuclear antigen protein content (P < .05) in human VSMCs. Confocal microscopy of both live and fixed cells showed modest oligonucleotide uptake that was primarily nuclear. Surprisingly, cationic liposomes did not enhance nuclear uptake but led to extensive, punctated cytoplasmic loading without an enhanced antisense effect. Oligonucleotides incubated with human coronary atherosclerosis fragments associated with cells within 1 hour, despite the presence of abundant extracellular matrix. S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated 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.

Tyrosine 114 is essential for the trimeric structure and the functional activities of human proliferating cell nuclear antigen.

In order to study the effect of trimerization of proliferating cell nuclear antigen (PCNA) on its interaction with DNA polymerase (pol) delta and its loading onto DNA by replication factor C (RF-C) we have mutated a single tyrosine residue located at the subunit interface (Tyr114) to alanine. This mutation (Y114A) had a profound effect on PCNA, since it completely abolished trimer formation as seen by glycerol gradient sedimentation and native gel electrophoresis. Furthermore, the mutant protein was unable to stimulate DNA synthesis by pol delta and did not compete effectively with wild-type PCNA for pol delta, although it was able to oligomerize and could to some extent interact with subunits of functionally active PCNA. We thus conclude that PCNA molecules that are not part of a circular trimeric complex cannot interact with the pol delta core. furthermore, the mutant protein could not be loaded onto DNA by RF-C and did not compete with wild-type PCNA for loading onto DNA, indicating that PCNA trimerization may also be a prerequisite for its recognition by RF-C. The adverse effects caused by this single mutation suggest that trimerization of PCNA is essential for the monomers to keep their overall structure and that the structural changes imposed by trimerization are important for interaction with other proteins.

Stimulation of DNA Polymerase γ Activity by Proliferating Cell Nuclear Antigen

DNA polymerase was partially purified from mitochondrial extracts of rat liver by phosphocellulose, DEAE-cellulose, heparin-Sepharose CL-6B and DNA-agarose column chromatography. By these purification steps, DNA polymerase and proliferating cell nuclear antigen (PCNA) were completely separated at the step of heparin-Sepharose CL-6B column chromatography. The isolated DNA polymerase was inhibited by ddTTP, but not by aphidicolin. The enzyme sedimented at about 8 S on 5-20 % analytical sucrose density gradient centrifugation. These data showed that the DNA polymerase isolated from mitochondria is γ in type. After the separation of DNA polymerase γ and PCNA, the two fractions were remixed and DNA polymerase γ activity was measured. DNA polymerase γ activity was stimulated about three-fold or more in the presence of the PCNA fraction. This stimulation was inhibited by the addition of anti-PCNA rabbit IgG2a. In addition, highly purified human recombinant PCNA stimulated the DNA polymerase γ activity. These results indicate that DNA polymerase γ, like DNA polymerase δ, is activated by PCNA.

Structure-Function Relationship of the Eukaryotic DNA Replication Factor, Proliferating Cell Nuclear Antigen

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase delta (pol delta). Due to the functional and structural similarity with the beta-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied 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 caused 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 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 which are exposed on one of the side surface 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 which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.

Expression and Physicochemical Characterization of Human Proliferating Cell Nuclear Antigen

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

A new method for phenotyping proliferating cell nuclear antigen positive cells using flow cytometry: implications for analysis of the immune response in vivo

The incorporation of radioactive nucleotides into newly synthesized DNA has been established as a standard method for the detection of proliferation in eucaryotic cells. Unfortunately the use of this method makes it harder to obtain information on the phenotype of proliferating cells in mixed cell populations. For this reason we established a flow-cytometric approach employing a monoclonal antibody specific for murine as well as human proliferating cell nuclear antigen (PCNA) and a double labeling technique for detection of cell membrane-expressed phenotypic markers. The efficiency of this immunostaining procedure was confirmed by simultaneous and highly specific detection of PCNA in nuclear structures as well as cell membrane-expressed antigens using cytological techniques. In vitro experiments with mitogen- and alloantigen-stimulated murine lymph node cells (LNC) and human peripheral blood mononuclear leukocytes (PBML) revealed a good correlation of total [ 3H]thymidine incorporation into DNA and expression of PCNA. For the analysis of proliferating cells activated in vivo the method was employed to evaluate the local lymph node assay which assesses the allergenicity of small chemicals. LNC prepared from the cervical lymph nodes of mice treated on 4 consecutive days with sensitizing concentrations of the contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic acid and SLS in comparison to the solvent control showed a dramatic increase in the total amount of proliferating cells for contact allergen-treated animals in comparison to the solvent control and irritant-treated mice. In addition a detailed phenotyping of the proliferating cell populations was possible. This approach offers an easy to perform, non-radioactive method for the assessment of proliferation of murine as well as human leukocytes in vitro and especially in vivo and will be of great advantage for situations where the phenotype of proliferating cellular subsets in heterogeneous populations is of interest.

Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter.

The proliferating cell nuclear antigen (PCNA) is an adenovirus E1A-inducible factor that is intimately linked to the processes of DNA replication and cell cycle regulation. Previously, we defined a novel cis-acting element, the PCNA E1A-responsive element (PERE), that confers induction by the E1A 243R oncoprotein upon the human PCNA promoter. To better understand the regulation of PCNA expression by E1A 243R, we have identified cellular transcription factors that associate with the PERE. In electrophoretic mobility shift assays, the PERE formed three major complexes (P1, P2 and P3) with proteins in nuclear extracts from HeLa or 293 cells. Formation of complexes P2 and P3, which correlates with PCNA promoter activity in vivo, requires the activating transcription factor (ATF) binding site found within the PERE [Labrie et al. (1993) Mol. Cell. Biol., 13, 1697-1707]. Antibody interference experiments and mobility shift assays performed with in vitro-synthesized protein indicated that the transcription factor ATF-1 is a major component of these complexes. Similar assays demonstrated that the hepatitis B virus enhancer-associated protein RFX1 constitutes a major component of the P1 complex. In addition, we examined the binding of proteins to the minimal E1A-responsive promoter to identify other factors important for transcription from the PCNA promoter. Mobility shift assays revealed that a fragment encompassing the region from -87 to +62 relative to the transcription initiation site forms at least five complexes, EH1-EH5, with HeLa cell nuclear extracts. The transcription factor YY1 associates with the initiator element of the PCNA promoter. The identification of these transcription factors will allow their roles in the activation of PCNA by E1A to be evaluated.

Molecular cloning of a maize cDNA clone encoding a putative proliferating cell nuclear antigen

We report the isolation and sequence of a maize cDNA clone which encodes a protein homologous to proliferating cell nuclear antigen (PCNA). The deduced amino acid sequence predicts a protein of 263 amino acids in length. The amino acid sequence shares 62% identity with the human PCNA and 95% identity with the rice homologue of PCNA.

Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic function and autoantibody recognition.

The proliferating cell nuclear Ag (PCNA) is a DNA replication factor postulated to function as a sliding clamp around DNA. PCNA is also a target for autoimmunity in systemic lupus erythematosus. The autoantigenicity of PCNA is highly conformation-dependent, and reaction with most anti-PCNA sera requires a nearly full-length PCNA molecule. Here we describe the use of gel filtration and glycerol gradient sedimentation to analyze the native structure and size of PCNA. PCNA from three sources was studied (PCNA from HeLa cells, PCNA purified after its overexpression in bacteria, and PCNA produced in the wheat germ cell-free translation system) as well as mutant forms of PCNA translated in vitro. In each case, full-length PCNA behaved as a trimer. Analysis of mutant proteins revealed a correlation between the trimeric form and binding to the common type of human anti-PCNA autoantibody, suggesting that the Abs are specific for the active form of the protein. These findings are consistent with the idea that autoantibodies are generated as a response to native Ag and provide experimental support for the hypothesis that PCNA serves its processive function in DNA replication as a trimeric ring structure.

Expression of functional proliferating-cell nuclear antigen from rice (Oryza sativa) in Escherichia coli. Activity in association with human DNA polymerase delta.

Proliferating-cell nuclear antigen (PCNA), the auxiliary protein for DNA polymerase delta, is one of the key factors for both PCNA-dependent DNA synthesis and cell-cycle progression. Plant PCNA genes have previously been cloned from rice, carrot, tobacco, and soybean cells by screening the cDNA libraries using similarity to the human or rat PCNA genes. We subcloned the relevant gene from the rice PCNA cDNA into an Escherichia coli expression vector pMAL, and the PCNA protein was expressed in the bacteria in the form of a fusion protein (70 kDa) with maltose-binding protein (MBP). Monoclonal antibody against human PCNA reacted with both purified fusion protein and a 32-kDa fragment, resulting from restriction protease (factor Xa) digestion of the fusion protein. The N-terminal amino acid sequence of the 32-kDa fragment was identical to that of rice PCNA sequence. Rice PCNA fusion protein was found to stimulate DNA synthesis catalyzed by DNA polymerase delta from human cells (although much less effectively), while having no effect on DNA polymerase alpha activity. The results indicate that plant PCNA functions as one of the cofactors of DNA synthesis as is the case with other eukaryotes.