DNA Polymerase Alpha Activity Research Articles

Effects of 12‐O‐Tetradecanoyl Phorbol‐13‐Acetate (TPA) on Rat Thymocytes

The short-term incubation of rat thymocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a significant increase in sialyltransferase (S-T) activity and a decrease in terminal deoxyribonucleotidyl transferase (TdT) activity. The ratio of peanut agglutinin (PNA)-positive cells and of TdT-positive cells in TPA-treated cells also decreased. However, TPA had no significant effects on the viability, morphology, DNA synthesis, and DNA polymerase alpha activity of the cells. More marked changes were observed by incubating a non-T, non-B human lymphoid leukaemia cell line with TPA. Similar findings were also noted in TPA-treated mouse thymocytes. These changes may represent an aspect of TPA-induced differentiation of murine thymocytes.

DNA polymerase alpha activity is not affected by protein kinases or alkaline phosphatase

Recent studies with crude or partially purified cell extracts have suggested that DNA polymerase α activity may be regulated by enzymatic phosphorylation. To further investigate these findings, we have examined the effects of protein kinases and phosphatases on highly purified DNA polymerase α from mouse cells. Incubation of DNA polymerase α with a variety of protein kinases, including protein kinase C, had no effect on polymerase activity. In addition, treatment of the polymerase with soluble calf intestinal alkaline phosphatase had no effect on DNA polymerase α activity, further indicating that phosphorylation does not have a direct role in modulating polymerase activity. In contrast, incubation of DNA polymerase α with calf intestinal alkaline phosphatase crosslinked to agarose beads resulted in a time dependent disappearance of polymerase activity. This loss of DNA polymerase activity was dependent on phosphatase activity, as the alkaline phosphatase inhibitors, potassium phosphate or levamisole, prevented the loss of polymerase activity in the presence of the beaded phosphatase. The loss of DNA polymerase α activity following beaded phosphatase treatment was not a general phenomena as the large fragment of Escherichia coli DNA polymerase 1, T4 DNA polymerase or mouse primase were not affected by similar treatment. The decreased DNA polymerase activity following incubation with phosphatase beads correlated with the binding of the DNA polymerase polypeptides, p185 and p68, to the agarose beads and this binding could not be reversed by either 150 mM potassium chloride or sodium sulfate. The binding of the polymerase to the agarose beads was dependent on the phosphatase activity, as the polymerase could be first treated with soluble calf intestinal phosphatase and subsequently bound to added Sepharose 4B beads. Surprisingly, Sepharose CL4B, a highly desulfated agarose preparation, did not bind the phosphatase-treated polymerase suggesting that sulfated polysaccharides are required for polymerase binding. The physiological correlate of this binding is unknown, but it has been reported that sulfated polysaccharides exist in a variety of intracellular compartments. It would be interesting to speculate that phosphorylation controls the intracellular compartmentalization of DNA polymerase α.

Interaction of Hyperthermia and Radiation in Tolerant and Nontolerant HeLa S3 cells: Role of DNA Polymerase Inactivation

The activities of DNA polymerase alpha and beta were measured in tolerant and nontolerant HeLa S3 suspension cells. The heat-inactivation of the enzymes and their recovery when cells were incubated at 37 degrees C after the heat challenge was compared to the synergistic action of heat and radiation and its disappearance at the level of cell survival. Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 37 degrees C was allowed between the two treatments. For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells. The results show that the activities of DNA polymerase alpha and beta do not always correlate with the extent of heat radiosensitization. It is concluded that heat inactivation of these enzymes may not be taken as a general cause for the synergistic effect of hyperthermia and radiation. As an alternative mechanism, changes in nuclear protein binding due to cellular heating are suggested, since these correlate well with effects observed for radiosensitization under different experimental conditions, including the use of thermotolerant cells.

The effect of medroxyprogesterone acetate on aromatase and DNA polymerase α activities in breast tumours

The effect of treatment with the progestogen medroxyprogesterone acetate (MPA) on the peripheral conversion of androstenedione to oestrone and tumour aromatase activity has been examined in post-menopausal women with advanced breast cancer. In addition to being a potent progestational compound, MPA also possesses glucocorticoid properties and glucocorticoids have been shown to stimulate in vitro aromatase activity. While some evidence was obtained of an increase in aromatase activity in tumour tissue after treatment with MPA, peripheral conversion of androstenedione to oestrone was similar when measured before (2.12 +/- 0.67%) and after (1.89 +/- 0.16%) treatment. DNA polymerase alpha activity, measured as a marker of cellular proliferation, decreased from 331 +/- 145 to 156 +/- 93 pmol thymidine triphosphate (TTP)/mg protein per h (P less than 0.02) in tumour samples examined before and after treatment. It is concluded that treatment with high doses of MPA has no effect on the peripheral conversion of androstenedione to oestrone but results in a significant reduction in tumour DNA polymerase alpha activity.

Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase.

To evaluate the relative contributions of DNA polymerase alpha and DNA polymerase delta in chromosome replication during the S phase of the cell cycle, we have used the permeable cell system for replication as a functional assay. We carried out the analysis of DNA polymerases both in quiescent cells stimulated to proliferate and progress through the cell cycle (monolayers) and in actively growing cells separated into progressive stages of the cell cycle by centrifugal elutriation (suspension cultures). DNA polymerase alpha was measured by using the inhibitor butylphenyl dGTP at low concentrations. Using several inhibitors such as aphidicolin, ddTTP and butylphenyl dGTP, we found that DNA polymerase alpha and delta activity were coordinately increased during S phase and declined at the end. However, DNA polymerase delta was performing about 80% of the total replication and DNA polymerase alpha performed only 20%. This high ratio of DNA polymerase delta to DNA polymerase alpha replication activity was maintained throughout S phase in two entirely different experimental approaches.

In vitro and in vivo cytogenetic studies of three β-lactam antibiotics (penicillin VK, ampicillin and carbenicillin)

The in vitro and in vivo clastogenic potential of three beta-lactam antibiotics, ampicillin, carbenicillin and penicillin VK, was investigated using cultured human lymphocytes and the rat micronucleus test. Neither ampicillin nor carbenicillin induced significant increases in chromosome damage in vitro up to test concentrations of 10 mg/ml. These results contrast with other published studies on these compounds. Both drugs were also inactive in vivo in the rat micronucleus test, using single- or double-dosing regimens (ampicillin 5 g/kg orally; carbenicillin 500 mg/kg i.m., either dosed once 30 h before marrow preparation, or dosed twice 48 and 24 h before marrow preparation). In vitro, penicillin VK induced a dose-related increase in chromosome and chromatid gaps and breaks, down to concentrations of 1.25 mg/ml. It is likely that the increase in aberration frequency was partly the result of exposing the cells to increased K+ ion concentration, as similar results were obtained when potassium chloride was evaluated over the same molar concentration range. However, the occurrence of 'ion-mediated' clastogenic effects as reported by other workers, does not fully account for the positive effects obtained with this compound, as clastogenic effects were also observed with penicillin V in this test system at similar test concentrations. It is known that exposure of mammalian cells to extremely high concentrations of beta-lactams can affect DNA polymerase alpha activity. An inhibitory effect upon DNA polymerase alpha resulting in a breakdown in the structural integrity of the chromosomes, is suggested as an additional mechanism of action for penicillin VK.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site.

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.

Herpes simplex virus replication in the presence of DNA polymerase alpha inhibitors

2-( p- n-butylanilino)deoxyadenosine (BuAdA), and N-2-( p- n-butylphenyl)deoxyguanosine (BuPdG), selective inhibitors of mammalian DNA polymerase alpha, were added to BHK-21(C13) cell cultures infected with herpes simplex virus type 1 (HSV-1) strain 17 syn +. Infectious virus production decreased significantly in the presence of the inhibitor at concentrations varying from 1 nM to 100 μM. BuPdG was more effective than BuAdA at all concentrations tested, while it inhibited virus yield as much as BuAdA when CVG2, a thymidine kinase deficient (TK-) HSV-1, was employed. HSV DNA synthesis, determined by quantitation of CsCl separated DNA peaks, was inhibited by each compound. BuPdG inhibited viral DNA replication more than BuAdA, while the effect on cell DNA synthesis was the same as that of BuAdA. CVG2 DNA replication was inhibited to the same level by BuAdA as by BuPdG. These results indicate that HSV DNA replication is partially dependent on cell DNA polymerase alpha activity, and that the greater effect of BuPdG on viral replication may be ascribed to its action on HSV thymidine kinase.

Differential inhibition of various mammalian DNA polymerase activities by ammonium 21‐tungsto‐9‐antimoniate (HPA23)

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.

Changes of enzymes involved in DNA synthesis in the testes of cryptorchid rats

The activities of DNA polymerase alpha (EC 2.7.7.7) and topoisomerase I did not fluctuate up to 7 days after surgery to induce cryptorchidism and showed no significant difference from those in control testes (sham-operated). In contrast, the activity of DNA polymerase beta decreased by 43% at 5 days (P less than 0.01) and by 47% at 7 days (P less than 0.001). The activity of DNA polymerase gamma also decreased by 46% at 3 days (P less than 0.02) and by 78% at 7 days (P less than 0.01) after surgery. The amount of mRNA for DNA polymerase beta decreased in parallel with enzyme activity. Since the sensitivity to heat inactivation of testicular DNA polymerase beta was exactly the same as that from liver, the decrease in DNA polymerase beta activity may be, at least in part, due to reduced biosynthesis of enzyme protein. The morphological changes in cryptorchid testes suggested that the decrease in DNA polymerase beta and gamma activities might be related to the deleterious effects of elevated temperature on spermatogenesis.

Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.

DNA polymerase alpha activity in mitogen-activated lymphocytes

DNA polymerase alpha isolated from adult-derived lymphocytes was separated into isozyme forms with low (A1) and high (A2) specific activity. In quiescent lymphocytes only A1 was detected, while mitogen-stimulated lymphocytes contained both A1 and A2 enzyme. Polymerase alpha A1, but not A2, interacted with phosphatidylinositol, ATP, and phosphatidylinositol kinase to yield an activated enzyme with increased affinity of binding to DNA. Mitogen-stimulated lymphocytes showed increased enzyme protein and total activity for both A1 and A2, but, when pre-treated with cycloheximide, exhibited an apparent increase in A2 specific activity with no increase in activity for A1 polymerase. These data suggest that mitogen stimulation of lymphocytes increased total DNA polymerase alpha activity by the phosphoinositide-related activation of polymerase alpha A1 to an A2-like form and by initiating de-novo synthesis of polymerase alpha A2.

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.

Evidence for a reduction of the replicative activity of matrix-bound DNA polymerase alpha following treatment with phospholipase C

Since phospholipids have been suggested to play some role in the molecular organisation of the nuclear matrix, the effect of their removal by means of phospholipase C has been investigated in regenerating rat liver nuclear matrix. The matrix-bound DNA polymerase alpha shows an almost complete loss of activity following the digestion with phospholipase C. Since the polymerase activity is restored by adding exogenous DNA, we suggest that the effect is due to the removal of matrix residual DNA, which is in some way linked to the nuclear matrix by means of hydrophobic interactions.

Regulation of deoxyribonucleic acid polymerase activity in uterine luminal epithelium after multiple doses of estrogen.

Estradiol (E2) exerts both inhibitory and stimulatory effects on DNA synthesis in the rat uterine luminal epithelium (LE). This inhibitory effect is due to a shift in the time course of DNA synthesis, i.e. in animals receiving a single injection of E2, a peak of DNA synthesis occurs 24 h after treatment, but in animals receiving multiple injections of E2, DNA synthesis is suppressed until 10-12 h after hormone treatment ceases. In these previous studies LE DNA synthesis was assessed by measuring tritiated thymidine incorporation. In the present study, we sought to determine if the molecular basis for this decrease in DNA synthesis was due to a suppression of DNA polymerase activity in LE nuclei. Animals receiving a single injection of E2 exhibit a peak of nuclear DNA polymerase activity 20-24 h later. Animals receiving multiple injections of E2 (0, 12, 15, and 18 h) show more than a 50% decrease in DNA polymerase activity at 20-24 h, due to a shift in the maximum increase in enzyme activity to 32-36 h after the initial treatment. The observed differences between these groups are not due to different levels of DNase activity or different degrees of leakage of the nuclear enzyme. The observed enzyme activity is due to DNA polymerase-alpha, since it requires ATP as well as deoxyribonucleoside triphosphates, and is aphidicolin sensitive. These results indicate that the inhibitory effect of E2 on LE DNA synthesis is due at least in part to a suppression of nuclear DNA polymerase-alpha activity.

Identification and subcellular localization of the polypeptide for chick DNA primase with a specific monoclonal antibody.

Polypeptides responsible for activities of chick embryo DNA primase and DNA polymerase alpha were identified using monoclonal antibodies specific to these two enzymes. The 4-8H antibody neutralized DNA polymerase alpha activity measured on activated DNA template and also ribonucleoside triphosphate-dependent DNA synthesis on single-stranded DNA template (DNA primase-DNA polymerase alpha combined activity) to a partial extent (about 30%), but did not affect DNA primase activity. The 4-2D antibody, although it did not affect DNA polymerase alpha activity, did neutralize both DNA primase activity and DNA primase-DNA polymerase alpha combined activity extensively (up to 70%). Immunoblotting analysis of the DNA primase-DNA polymerase alpha complex showed that 4-2D and 4-8H antibodies recognize 60-kDa and 160-180-kDa polypeptides, respectively. An immunoaffinity column made of either of these antibodies retained DNA primase-DNA polymerase alpha complex. When the enzyme was eluted from the 4-8H column with alkaline solution, DNA primase was eluted prior to DNA polymerase alpha. In the case of 4-2D antibody column chromatography, the elution order of two enzymes was reversed. Results indicate that two enzymes in the complex which was retained in the antibody column were dissociated by lower alkaline pH than that dissociated the antigenic enzymes from the corresponding antibodies. In both cases, the fractions with DNA primase activity contained exclusively 60-kDa polypeptide, while those with DNA polymerase alpha contained 160-180-kDa polypeptides. Thus, DNA primase resided in 60-kDa polypeptide and was recognized by 4-2D antibody while DNA polymerase alpha resided in 160-180-kDa polypeptides and was recognized by 4-8H antibody. Immunofluorescence made with the DNA primase-specific 4-2D antibody as well as with 4-8H antibody appeared in granular structures which were tightly bound to the nuclear matrix. These nuclear fluorescences were much reduced in quiescent cells. Furthermore, since the fluorescence made by these antibodies was induced by adding serum to the quiescent cells in serum-deprived cultures, the expression of DNA primase and its organization in the structures on the nuclear matrix are regulated in correlation to the proliferating stage of cells, as observed with DNA polymerase alpha.

C-myc protein and DNA replication: separation of c-myc antibodies from an inhibitor of DNA synthesis.

Antibodies against human c-myc protein have been reported to inhibit DNA polymerase activity and endogenous DNA synthesis in isolated nuclei, suggesting a role for c-myc in DNA replication. Using the same antibody preparations, we observed equivalent inhibition of simian virus 40 DNA replication and DNA polymerase alpha and delta activities in vitro, as well as inhibition of DNA synthesis in isolated nuclei. However, the c-myc antibodies could be completely separated from the DNA synthesis inhibition activity. c-myc antibodies prepared in other laboratories also did not interfere with initiation of simian virus 40 DNA replication, DNA synthesis at replication forks, or DNA polymerase alpha or delta activity. Therefore, the previously reported inhibition of DNA synthesis by some antibody preparations resulted from the presence of an unidentified inhibitor of DNA polymerases alpha and delta and not from the action of c-myc antibodies.

C-myc protein and DNA replication: separation of c-myc antibodies from an inhibitor of DNA synthesis.

Antibodies against human c-myc protein have been reported to inhibit DNA polymerase activity and endogenous DNA synthesis in isolated nuclei, suggesting a role for c-myc in DNA replication. Using the same antibody preparations, we observed equivalent inhibition of simian virus 40 DNA replication and DNA polymerase alpha and delta activities in vitro, as well as inhibition of DNA synthesis in isolated nuclei. However, the c-myc antibodies could be completely separated from the DNA synthesis inhibition activity. c-myc antibodies prepared in other laboratories also did not interfere with initiation of simian virus 40 DNA replication, DNA synthesis at replication forks, or DNA polymerase alpha or delta activity. Therefore, the previously reported inhibition of DNA synthesis by some antibody preparations resulted from the presence of an unidentified inhibitor of DNA polymerases alpha and delta and not from the action of c-myc antibodies.

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)