Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.     

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.     

Inhibition of nuclear DNA synthesis by an autoantibody to proliferating cell nuclear antigen/cyclin

Proliferating cell nuclear antigen (PCNA) is expressed in the nuclei of proliferating cells, but is not detected in resting cells. The kinetics of PCNA expression suggest that it is associated with a phase preceding active DNA synthesis. DNA synthesis is under cytoplasmic control, and there is a cytoplasmic protein, ADR (activator of DNA replication), that induces DNA synthesis in isolated quiescent nuclei. We now report that a human antibody preparation monospecific for PCNA, but not two monoclonal antibodies directed against different epitopes on PCNA, can inhibit the ability of ADR to induce DNA synthesis in isolated quiescent nuclei. This effect is not due to inhibition of DNA polymerase alpha activity. Thus, the anti-PCNA antibody exerts its effect either by directly influencing the initial interaction of ADR with the nucleus, or by inhibiting subsequent synthetic events.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.     

Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen

DNA polymerase (Pol) λ is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol λ has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol λ has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol λ. Pol λ is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol λ was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol λ was decreased by its interaction with PCNA. Finally, Pol λ is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.     

Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2-kinase in mouse T-lymphoma cells

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.     

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

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.     

Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin

We have previously reported the purification of yeast analogs to mammalian DNA polymerase delta and proliferating-cell nuclear antigen (PCNA)/cyclin: DNA polymerase III and yeast PCNA, respectively. Through the use of gel-filtration chromatography, we have studied the interaction of the model template-primer system poly(dA).(dT)16 (40:1) with yeast DNA polymerase III and with PCNAs. Yeast DNA polymerase III binds to the DNA in the absence of yeast PCNA/cyclin, but comigration of either yeast or calf thymus PCNA/cyclin with the DNA requires the additional presence of yeast DNA polymerase III. We could also isolate a DNA-calf thymus DNA polymerase delta-calf thymus PCNA/cyclin complex. From these data, we propose that PCNA/cyclin is involved not in the binding step of the polymerase to the template-primer, but in the elongation step. The 3'----5' exonuclease associated with yeast DNA polymerase III acts in a distributive manner on poly(dA).(pT)16, and dissociates from the DNA when addition of dTTP allows switching from the exonuclease to the polymerase mode. Addition of PCNA/cyclin had no effect on these activities.     

Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.     

Arabidopsis thaliana proliferating cell nuclear antigen has several potential sumoylation sites

Proliferating cell nuclear antigen (PCNA) is post-translationally modified in yeast and animal cells. Major studies carried out in the last decade have focused on the role of sumoylated and ubiquitinated PCNA. Using different approaches, an interaction between plant PCNA and SUMO both in vivo and in bacteria has been demonstrated for the first time. In addition, identical sumoylation patterns for both AtPCNA1 and 2 were observed in bacteria. The plant PCNA sumoylation pattern has been shown to differ significantly from that of Saccharomyces cerevisiae. This result contrasts with a common opinion based on previous structural analysis of yeast, human, and plant PCNAs, which treats PCNA as a highly conserved protein even between species. Analyses of AtPCNA post-translational modifications using different SUMO proteins (SUMO1, 2, 3, and 5) revealed similar modification patterns for each tested SUMO protein. Potential target lysine residues that might be sumoylated in vivo were identified on the basis of in bacteria AtPCNA mutational analyses. Taken together, these results clearly show that plant PCNA is post-translationally modified in bacteria and may be sumoylated in a plant cell at various sites. These data open up important new perspectives for further detailed studies on the role of PCNA sumoylation in plant cells.     

Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.