Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).     

Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex.

The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta).proliferating cell nuclear antigen (PCNA).DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin. streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin. streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.     

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.     

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.     

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.     

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.     

DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA

In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer.     

Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen

A large form of DNA polymerase delta from HeLa cells was recently purified in this laboratory as a factor required for conservative DNA synthesis in a reconstituted system utilizing UV-irradiated permeabilized human diploid fibroblasts (Nishida, C., Reinhard, P., and Linn, S. (1988) J. Biol. Chem. 263, 501-510). We have now purified this form of the enzyme utilizing its polymerase activity and further characterized it. The enzyme activity sediments at 11.1 S in low salt and 6.8 S in high salt. In both cases, activity cosediments with the major visible peptide displayed by sodium dodecyl sulfate-polyacrylamide gels which has an Mr of 215,000. This value is consistent with the molecular mass calculated from the sedimentation coefficient and gel filtration behavior in high salt. In low salt the apparent molecular mass was approximately double. The enzyme prefers poly(dA).oligo(dT) as template/primer in low salt, with which it has a processivity of several thousand nucleotides in 1 mM MgCl2. At isotonic KCl or potassium phosphate concentrations, the preferred template/primer is activated DNA. Proliferating cell nuclear antigen, also characterized as a DNA polymerase delta auxiliary protein, does not increase the activity of this preparation of the enzyme. An antibody to the proliferating cell nuclear antigen has no inhibitory effect, nor is it able to recognize any peptides in immunoblots of purified enzyme fractions. Under polymerizing conditions, the enzyme removes mismatched, but not matched nucleotides from the 3' terminus of oligo(dT) annealed to poly(dA) suggesting a proofreading function. The properties of this form of DNA polymerase delta distinguish it from other preparations reported in the literature.     

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.     

Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase delta.

The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol delta is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol delta interacts with PCNA.     

Autoantibody to the proliferating cell nuclear antigen neutralizes the activity of the auxiliary protein for DNA polymerase delta.

Two murine monoclonal antibodies to the proliferating cell nuclear antigen (PCNA), a rabbit anti-N-terminal peptide antibody and human auto-antibody to PCNA reacted with the auxiliary protein for DNA polymerase delta from fetal calf thymus following SDS-polyacrylamide gel electrophoresis, confirming the identity of PCNA and the auxiliary protein. Undenatured auxiliary protein was immunoprecipitated by the human autoantibody, but not by the monoclonal antibodies, which were raised to SDS-denatured PCNA, nor by the anti-N-terminal peptide antibody, suggesting that the epitopes recognized by both the monoclonal antibodies and the anti-peptide antibody are not exposed in the native protein. The human anti-PCNA autoantibody neutralized the activity of the auxiliary protein for DNA polymerase delta, but did not inhibit the activity of pol delta itself. The ability of pol delta to utilize template/primers containing long stretches of single-stranded template was inhibited by the anti-PCNA autoantibody, whereas the activity of pol alpha on such templates was not affected, confirming the specificity of the auxiliary protein for pol delta. The ability of PCNA, a cell cycle-regulated protein, to regulate the activity of pol delta suggests a central role for pol delta in cellular DNA replication.     

Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.     

A novel PDIP1-related protein, KCTD10, that interacts with proliferating cell nuclear antigen and DNA polymerase delta

Rat potassium channel tetramerisation domain-containing 10 (KCTD10) gene was cloned and identified as a novel member of polymerase delta-interacting protein 1 (PDIP1) gene family. Rat KCTD10 is highly expressed in lung and moderately expressed in heart and testis. KCTD10 shares significant similarity in amino acid sequence to PDIP1 and can interact with the small subunit of DNA polymerase delta and PCNA as PDIP1 does. Like PDIP1, the expression of KCTD10 gene can be induced by TNF-alpha in NIH3T3 cells.     

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrated with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacted with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis revealed that the putative Drosophila PCNA was highly homologous to human PCNA. Of the first 22 amino acids, 16 were identical, and 4 of the remaining 6 were changed conservatively. Results of total amino acid analysis were also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrated that Drosophila PCNA could substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-reacted with human PCNA and were able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ. These antibodies thus promise to be useful probes for the study of cell proliferation in this rapidly developing organism.     

Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.     

Identification of a novel protein,PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen

The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.     

Calf thymus DNA helicase F, a replication protein A copurifying enzyme.

A DNA helicase from calf thymus, called DNA helicase F, copurified with replication protein A through several steps of purification including DEAE-Sephacel, hydroxyapatite and single stranded DNA cellulose. It is finally separated from replication protein A on FPLC Mono Q where the DNA helicase elutes after replication protein A. Characterization of the DNA helicase F by affinity labeling with [alpha 32P]ATP indicated that the enzyme has a catalytic subunit of 72 kDa. Gel filtration experiments suggested that DNA helicase F can exist both in a monomeric and an oligomeric form. The enzyme unwinds DNA in the 5'-->3' direction in relation to the strand it binds. All eight deoxyribonucleoside- and ribonucleosidetriphosphates could serve as an energy source. Testing a variety of DNA/DNA substrates demonstrated that the DNA helicase F preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail. However, replication protein A allowed the DNA helicase to unwind much longer DNA substrates of up to 400 bases, indicating that the copurification of replication protein A with the DNA helicase F might be of functional relevance.