Multiple Forms of DNA Polymerase in Mouse Myeloma

Abstract

Five distinct forms of DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) were separated from extracts of mouse myeloma MOPC-104E using a fractionation procedure based upon sequential iop-exchange column chromatography. The The existence of multiple forms of DNA polymerase in eukaryotic tissues is well established (1-9). The extent of the multiplicity and nature of the enzymes involved are matters of current investigation. DNA polymerases that exhibited apparently novel template specificity were recently found in several tissues, including chicken embryo (10, 11), HeLa cells (12), rat liver (13), Xenopus oocyte (14), and mouse myeloma (15). A characteristic feature of these enzyme activities was that all were relatively inactive with natural DNA templates but were active with poly(rA)-containing templates; however, the relationship of these enzyme activities to previously studied DNA polymerases was not apparent. Enzymes corresponding to the well characterized 2.5-3.5S cellular DNA polymerase may have been responsible for the poly(rA)-directed activities, since this enzyme from bone marrow used poly(rA) as template with much higher efficiency than natural DNA (16). These observations prompted the current study, in which we attempted to determine the total number of both poly(rA)-directed and DNA-directed DNA polymerases in mouse myeloma (17). Hydroxyapatite Column Chromatography. Fractions from phosphocellulose (PC) columns containing DNA polymerase activities were pooled as indicated in Fig. 1. Protein that precipitated in the presence of 65%o saturated ammonium'sulfate was dissolved in a solution containing 50 mM Tris * HCI (pH 7.7), 50% (v/v) glycerol, 1 mM DTT, and 500 mMKCl and stored at -30°. A portion of this solution was dialyzed against 5 mM potassium phosphate buffer (KPi) in HA solution (500 mM KCl, 20% glycerol, 1 mM DTT, 1 mM EDTA, and 0.2% Nonidet P40), pH 6.4, and applied to a hydroxyapatite (fIypatite Clarkson Chem. Co.) column (0.8 X 15 cm); the column was developed using a 60-ml linear gradient of 5 mM to 100 mM KPi HA solution (pH 6.4-7.0 (Fig. 1) and the total amount of activity recovered was equal to a major portion of the DNA polymerase activity detected in the crude extract (Table 1). The position of elution from the PC column of each peak of activity was found to be reproducible to within i 10 mM KPi using extracts either from the same passage of myeloma or from a different passage of the tumor. Except in the flowthrough fractions, no common peaks of activity were observed in the PC chromatograms of DEAE-cellulose fractions D1

DNA Polymerases of Rat Liver

“…(dT),, as primertemplate was observed. Since a poly(rA)-dependent DNA polymerase enzyme (synthetic RNA-dependent or R-DNA polymerase) found in a wide variety of eukariotic cell types [12,29,33,54-561 was also discovered in soluble extract of rat liver [53,57], the above observed activity may be the result of contamination with RNA-dependent DNA polymerase. This activity has been reported to be separable from DNA-dependent DNA polymerase [53,57].…”

“…Incorporation in the absence of one or more deoxynucleotides with other highly purified mammalian DNA polymerases has been reported [12,16,28].…”

Partial purification and characterization of DNA polymerases from the cauliflower inflorescence.

“…Multiple forms of DNA polymerases have been detected in a number of eukaryotic organisms, and these enzymes have very different properties (Wallace et al 1971;Chang and Bollum 1971;Sedwick et al 1972;Matsukage et al 1974;Lynch et al 1975;Brakel and Blumenthal 1977;Weissbach 1977). Activity varies markedly depending on the developmental or physiological state of the cells (Chiu and Sung 1972;Benbow et al 1975;Ford et al 1975;Bertazzoni et al 1976;Spadari et al 1978).…”

The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum

“…All cells so far studied have shown more than one enzyme with DNA polymerase properties (Holmes & Johnston, 1975;. Further, it has been demonstrated that enzymic activities can be associated with subcellular structures such as nuclei (Bernard et al, 1974), mitochondria (Meyer & Simpson, 1970) and cytoplasmic membrane fractions (Matsukage et al, 1974). Moreover it has been established that in DNA-containing virions multiple activities of DNA-dependent DNA polymerase can be separated by ion-exchange chromatography (Lin et al, 1973).…”