Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.     

Viral polypeptides detected by a complement-dependent neutralizing murine monoclonal antibody to human cytomegalovirus.

Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000- and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000- and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000- and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35S]methionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfide-linked 130,000-Da polypeptides.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Detection of Epstein-Barr virus-associated antigens and DNA in salivary gland biopsies from patients with Sjogren's syndrome

Sjogren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. To determine whether Epstein-Barr virus (EBV) might play a role in the pathogenesis of this disorder, we used monoclonal antibodies and DNA probes to detect evidence of viral gene products and genomes in these patients' tissue biopsies and saliva. Cytoplasmic staining of epithelial cells (i.e., ductal and/or acinar cells) with monoclonal antibody against the EBV-encoded early antigen (EA-D) was noted in 8/14 salivary gland biopsies from SS patients. This antibody did not react with normal salivary glands or salivary gland tumors, nor with other tissues from SS patients. The reactive antigen in SS biopsies had a m.w. of 52,000 on the basis of immunoblotting experiments, similar to the EA-D antigen found in lymphoblastoid cells lytically infected with EBV. EBV DNA was detected in parotid biopsies from two SS patients in amounts ranging from 0.1 to 3 pg per 20 micrograms of cellular DNA. Southern blotting was used to demonstrate the reactivity with Bam V, Eco D, and Bam M probes. Parotid saliva samples from 8/20 SS patients contained EBV DNA detectable by slot blot hybridization. EBV DNA was not detected in saliva of age-matched controls, rheumatoid arthritis patients lacking sicca symptoms, or patients with benign parotid tumors. The presence of EBV in salivary gland and saliva samples was associated with clinically more severe SS, as manifested by extraglandular symptoms such as vasculitis, occurrence of "pseudolymphoma", and marked abnormalities in immunoglobulin levels. These results demonstrate an elevated content of EBV in salivary glands of SS patients, and suggest that this virus may play a role in pathogenesis.     

A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro

The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.     

Phosphorylation of the Epstein-Barr virus (EBV) DNA polymerase processivity factor EA-D by the EBV-encoded protein kinase and effects of the L-riboside benzimidazole 1263W94.

A member of the family of L-riboside benzimidazole compounds, 1263W94, was shown recently to inhibit replication of Epstein-Barr virus (EBV) (V. L. Zacny, E. Gershburg, M. G. Davis, K. K. Biron, and J. S. Pagano, J. Virol. 73:7271-7277, 1999). In the present report the effect of 1263W94 on the phosphorylation pattern of the EBV DNA polymerase processivity factor, EA-D, during viral reactivation in latently EBV-infected Akata cells is analyzed. This pattern specifically changes with progression of cytolytic infection. In the presence of 1263W94 the appearance of the hyperphosphorylated form of EA-D is mainly affected. Next, coexpression of the cloned EBV-encoded protein kinase (EBV PK), BGLF4, with EA-D demonstrated the ability of EBV PK to phosphorylate EA-D to its hyperphosphorylated form in transient assays. However, the phosphorylation of EA-D was not directly inhibited by 1263W94 in these coexpression assays. The results indicate that the EBV PK appears to be responsible for the hyperphosphorylation of EA-D, imply that the phosphorylation status of EA-D is important for viral replication, and suggest that 1263W94 acts at a level other than direct inhibition of EA-D phosphorylation by EBV PK.     

DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

Characterization of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha purified from calf thymus using monoclonal antibody.

Existence of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha was shown by production of a monoclonal anti-calf thymus 10S DNA polymerase alpha antibody secreted from a hybridoma line named 3H1. The antibody bound three polypeptides with Mr = 180,000, 56,000 and 32,000 in hydroxylapatite fraction of 10S DNA polymerase alpha by immunoblot. The antibody co-precipitated the polypeptides with the large polypeptide (Mr = 150,000-140,000) of 10S DNA polymerase alpha with the aid of second antibody. Among three polypeptides, the Mr = 56,000 polypeptide was co-purified with DNA polymerase alpha through DNA-cellulose chromatography and repeated sucrose rate-zonal centrifugations. The Mr = 56,000 polypeptide was still associated with 10S DNA polymerase alpha after second sucrose rate-zonal centrifugation, but the amount of it was reduced. The polypeptide was banded at pH 7.2-8.0 and displayed microheterogeneity in respect of isoelectric point by isoelectrofocusing with 7 M urea, and showed weak DNA-binding property after blotting onto a nitrocellulose. The antibody against the polypeptide precipitated DNA polymerase alpha from human, rat, and mouse, and Mr = 56,000 and 32,000 polypeptides were detected in these DNA polymerase alpha fractions by immunoblot. These results suggest that the polypeptide with Mr = 56,000 may take part in the DNA polymerase reaction.     

Specific immune serum to the Epstein-Barr virus DNA polymerase.

Epstein-Barr virus (EBV) DNA polymerase was released from phorbol ester-treated tamarin (Saguinus oedipus) cells (B95-8) and prepared for use as an antigen by sequential column chromatography with DEAE-Sephadex A-25, DEAE-cellulose, phosphocellulose, and single-stranded DNA cellulose. Proteins from single-stranded DNA cellulose with DNA polymerase activity in 100 mM ammonium sulfate were mixed with complete Freund adjuvant and injected intradermally into rats and rabbits. Immune sera that were screened for specific antibody by indirect immunofluorescence procedures reacted with approximately 3% of the cells in EBV-producer cultures (B95-8 and P3HR-1) but not with EBV genome-negative cells (BJAB). In functional enzyme assays, immune sera or the immunoglobulin fraction inhibited the activity of purified EBV DNA polymerase 90%. Inhibition of enzyme activity was not affected by absorption of immune sera with insoluble matrices of proteins prepared with tamarin and human cells which lacked the EBV genome. Cellular DNA polymerase alpha was not inhibited by immune sera to the EBV enzyme.     

Immunochemical studies of DNA polymerase delta: relationships with DNA polymerase alpha

A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides.     

Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells

Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently.     

Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans.

The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV-specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes.     

Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation

The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex. The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S. The calculated molecular weight of the reconstituted complex is 312,000. We infer that the stoichiometry of the complex is one of each subunit per complex. The complex has a prolate ellipsoid shape with an axial ratio of approximately 16. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody. While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits). The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact. However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity. The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit. The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.     

Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.     

Large polypeptides of 10S DNA polymerase alpha from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis

The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.     

Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.     

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA polymerases alpha and delta are immunologically and structurally distinct

The relationship between DNA polymerases alpha and delta are evaluated immunologically by monoclonal antibody specifically against DNA polymerase alpha and murine polyclonal antiserum against calf thymus DNA polymerase delta. DNA polymerases alpha and delta are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase delta and DNA polymerase alpha from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase delta and DNA polymerase alpha are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase delta from HeLa cells was also assessed. This PCNA-independent human polymerase delta was found to be immunologically unrelated to human polymerase alpha but to share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase delta.     

Monoclonal antibody specific for chicken DNA polymerase alpha associated with DNA primase

Four monoclonal antibodies against chicken DNA polymerase alpha were obtained from mouse hybridomas (see ref. 1). Two of them, 4-2D and 4-8H, recognized different epitopes of the DNA polymerase alpha-DNA primase complex as determined by a competitive enzyme-linked immunosorbent assay. Antibody 4-8H partially (about 30%) neutralized the combined activity of primase-DNA polymerase alpha as well as the DNA polymerase alpha activity. In contrast, antibody 4-2D did not neutralize DNA polymerase alpha activity, but neutralized the primase-DNA polymerase alpha activity extensively (up to 80%). Furthermore, although an immunoaffinity column made with 4-8H antibody retained virtually all of the DNA polymerase alpha with and without associated primase, a column made with 4-2D antibody did not bind DNA polymerase alpha without the primase, but retained the enzyme associated with the primase. These results indicate that 4-8H monoclonal antibody is specific for DNA polymerase alpha and 4-2D monoclonal antibody is specific for the primase or a special structure present in the primase-DNA polymerase alpha complex.