Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody.

Abstract

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...

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

“…To assess whether the Pol variant retains the ability to incorporate deoxynucleoside triphosphates (dNTPs), we took advantage of the selective increase in Pol catalytic activity in the presence of manganese to address its polymerization activity using an in-gel activity assay (18). Starting with a protein mixture, this technique enables the enzymatic activity of a given protein to be assayed following SDS-PAGE in the presence of activated DNA.…”

Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the aprt locus of Chinese hamster ovary cells.

“…No 3'--5' exonuclease was found to be associated with the replicative polymerase a complexes recovered by immunoaffinity chromatography (33,34). However, an exonuclease cosedimenting with polymerase a was recently reported (19), reminiscent of the associated proofreading subunit of the E. coli pol III (35).…”

Autoantibody to RNA polymerase I in scleroderma sera.

“…Further investigations will determine whether these additional polypeptides are part ofthe RNA polymerase I complex reflecting additional subunits or merely posttranslational modifications of previously described subunits. It is also conceivable that some of these additional polypeptides represent tightly bound factors of this enzyme that may be associated with initiation or termination of rRNA gene transcription (38), Recently, immunoprecipitation of intrinsically radiolabeled DNA polymerase a with a monoclonal antibody resulted in the identification of an additional catalytic component of this enzyme that had been previously unknown (39). It seems likely that the use of specific antibody for immunoprecipitation will also facilitate further characterization of RNA polymerase I.…”