Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster.

Abstract

A procedure has been devised for the purification of intact DNA polymerase a from early embryos of Drosophila melanogaster. The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000. These are related antigenically to the a (Mr 148,000), .l (Mr 58,000), and Y (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G. R., Boezi, J. A. & Lehman, I. R. (1979)J. Biol. Chem. 254, 9886-9892]. The-a subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo. As in the previous preparation, the a subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme. The ratio of primase to polymerase remains constant throughout the purification. Thus, the primase is very likely an integral component of the Drosophila DNA polymerase a. The purified DNA polymerase-primase contains no detectable endoor exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCI optima identical to those reported previously. In contrast, the Km for dTTP is 3.7 ,uM as compared with 17.5 ,uM for the previous enzyme. Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged.DNA polymerase a as purified from a variety of eukaryotes consists of two or more polypeptides. Several polymerases (1-7) have been isolated as multisubunit proteins consisting of a catalytic core with an approximate Mr of 150,000 in association with three or four smaller subunits whose Mrs range from 40,000 to 60,000. However, other polymerase preparations are lacking a high molecular weight subunit (8,9). Our recent studies (10) and those of others (11)(12)(13)(14) have shown that a DNA primase activity is associated with the a polymerase and may be part of the multisubunit molecule.Uncontrolled proteolysis during purification has prevented the unambiguous determination of the subunit structure of DNA polymerase a and the structure-function relationships of its individual polypeptides. That proteolysis may yield an active but highly degraded form of the enzyme has been clearly demonstrated both in Drosophila melanogaster embryos and in calf thymus (4,(15)(16)(17) were prepared for use as described (19). Leupeptin was purchased from the Peptide Institute (Minoh-shi, Japan). Aphidicolin was prepared as a stock solution at 1 mg/ ml in 40% ethanol/10% dimethyl sulfoxide. N-Ethylmaleiimide (Schwarz/Mann) was dissolved in water immediately before use.Antisera. Antisera directed against Drosophila DNA polymerase a and its isolated a and J subunits were prepared 'as described (18). Purified IgG was prepared by ammonium sulfate precipitation and DEAE-cellulose chromatography (20).Enzymes. Escherichia coli DNA polymerase I and E. coli RNA polymerase were the generous gifts of J. Kelly and J. Kaguni, respectively, of this department. Pancreatic DNase I was purchased from Worthington.Nucleic acids. Calf thymus DNA (grade A, Calbiochem) wa...

Four Deoxynucleoside Kinase Activities from Drosophila melanogaster Are Contained within a Single Monomeric Enzyme, a New Multifunctional Deoxynucleoside Kinase

“…dThd was a strong inhibitor of the phosphorylation of dCyd, dAdo, and dGuo with these data indicate that the four substrates are phosphorylated at the same site of the enzyme. The absence of a TK equivalent to the mammalian S-phase specific TK1 was unexpected, as other DNA replication associated enzymes previously have been isolated from Drosophila embryonic cells (25,26). Additionally, the absence of a dCK equivalent to the mammalian dCK should be noted.…”

Wanderings of a DNA Enzymologist: From DNA Polymerase to Viral Latency

“…They showed that Pol α consisted of four subunits; the largest, at 180 kDA, contained the DNA polymerase activity (42). Ron Conaway, a graduate student, then demonstrated that the most purified preparations of Pol α contained primase activity (primase had not yet been demonstrated in eukaryotes); he and Laurie Kaguni showed that the primase was associated with the two smaller of the four subunits (43). The association of primase with Pol α in Drosophila was quickly confirmed by several laboratories, in eukaryotes as diverse as baker's yeast and humans.…”

Function of DNA Polymerase α in a Replication Fork and its Putative Roles in Genomic Stability and Eukaryotic Evolution

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