An assay of adenosine(5′)tetraphospho(5′)adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 μM to 13 μM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1‐arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000‐fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 μM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 μM) in a “committed” but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 μM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature‐down‐shift experiments, using chick embryo cells infected with transformation‐defective temperature‐sensitive viral mutants(td‐ts), have shown that the expression of the transformed state at 35°C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a “second messenger” of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase α, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.
A monoclonal antibody against purified calf DNA polymerase a (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Most attempts to elucidate the subunit structure of DNA polymerase a (a-polymerase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) have relied upon NaDodSO4/polyacrylamide gel electrophoretic analysis of highly purified preparations. However, identification of enzyme subunits by this approach is complicated by the possibilities of both proteolytic degradation and elimination of important enzyme polypeptides during laborious isolation procedures. Clearly, additional methods, not involving laborious purification, are needed for the study of apolymerase polypeptides. Recently, "activity gel" analysis (1-3) of crude homogenates resulted in the identification of a Mr 110,000-120,000 a-polymerase catalytic polypeptide in a number of eukaryotic tissues; in addition, putative a-polymerase catalytic polypeptides also were detected at about Mr 70,000 (1-3). Nevertheless, use of the activity gel method is complicated by the fact that the detection efficiency of apolymerases is low, and some purified a-polymerases are completely unable to produce an activity signal (2). A promising immunological approach, based upon solid-phase immunobinding, was recently reported by Sauer and Lehman (4). Those workers observed a Mr 182,000 polypeptide in crude extracts of Drosophila embryo that cross-reacted with an antiserum raised against the Mr 148,000 polypeptide of purified Drosophila a-polymerase; generally, a-polymerase subunits of Mr > 160,000 had not been reported prior to this work. Lehman and co-workers (5, 6) subsequently showed that the Mr 182,000 polypeptide itself was capable of DNA polymerase catalytic activity.Studies of a-polymerases have been facilitated recently by the development of monoclonal antibodies to these enzymes. Kom and co-workers (7, 8) used a monoclonal antibody to KB cell a-polymerase to localize the enzyme by immunocytofluorescence, and this group and Wahl et al. used a monoclonal antibody to purify the enzyme by immunoaffinity chromatography (9, 10). Masaki et al. (11) found that a monoclonal antibody to calf a-polymerase could distinguish individual species of the enzyme in partially purified preparations from calf thymus, and Matsukage et al. (12) used a monoclonal antibody to chicken a-polymerase to study tissue-specific expression of the enzyme as a function of embryonic development. In the present study, we used a monoclonal antibody approach to elucidate components of mammalian cell lines that share immunological determinants with purified a-polymerase. Proteins in a crude soluble extract from growth-phase monkey cells were subjected to immunoprecipitation with one of our monoclonal antibodies to apolymerase. Immunoprecipitated polypeptides were electrophoresed in NaDodSO4/polyacrylamide gels and then examined for DNA polymerase activ...
Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electropho-
A sample of highly purified calf thymus alpha-polymerase contained an abundant 118,000 Mr polypeptide as well as five lower molecular weight polypeptides in the range of 54,000- to 64,000-Mr. This 118,000-Mr polypeptide was capable of DNA polymerase activity, as revealed by in situ assay after SDS-polyacrylamide gel electrophoresis. Tryptic peptide mapping indicated that the 118,000-Mr polypeptide shared extensive primary structure homology with 57,000-, 58,000- and 64,000-Mr polypeptides and some limited homology with 54,000- and 56,000-Mr polypeptides. This is the first evidence that lower and higher Mr polypeptides of purified calf thymus alpha-polymerase share sequence homology; these results are interpreted in the context of a model that predicts the existence of a common precursor with molecular weight greater than 140,000.
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