Nuclear Deoxyribonucleic Acid Polymerase

HeLa cell DNA polymerase .gamma.: further purification and properties of the enzyme

“…The extended purification of DNA polymerase from HeLa cells, free of oncorna virus particles, has yielded an enzyme preparation with a specific activity of 25 000 units/mg of proteins which is 70-fold greater than that previously reported (Spadari and Weissbach, 1974). This specific activity can be compared with that of the purified human a-polymerase (7281 units/mg; Sedwick et al, 1975) or to the purified human ßpolymerase which shows a specific activity of 6000-36 000 units/mg with a synthetic homopolymer template (Wang et al, 1974). The purified enzyme seems to exist as a high molecular weight structure which showed an estimated molecular weight of 160 000 (by sedimentation velocity), or 275 000-330 000 (by acrylamide gel electrophoresis or by Sephadex gel filtration).…”

Effect of manganese(2+) ions on the in vitro activity of human deoxyribonucleic acid polymerase .beta.

“…Poly(dA) and d(pT)j were from Collaborative Research. Oligonucleotides (dT)i6, (dT)5 §, [3H](dC)g.7 (14 170 cpm/pmol terminal dCMP residue), (dT)2jo, and (dT)2og-[3H](dT)5 (16 800 cpm/pmol terminal dTMP residue) were prepared as before (Wang et al, 1974) with terminal transferase, using d(pT)j as initiator. The average chain lengths of the oligonucleotide products were estimated as described by Richardson (1966).…”

“…[5,-32P]d(pT),-d(pT)-266 (27 600 cpm/pmol) was synthesized according to Lehman and Chien (1973). Homopolymer primer-templates were prepared as described (Wang et al, 1974). Micrococcal nuclease, spleen phosphodiesterase (SPH), bacterial alkaline phosphatase (BAPF), pancreatic DNase I (DPFF), and snake venom 1 Abbreviations used: Tris, tris(hydroxymethyl)aminomethane; PEI, polyethylenimine; EDTA, ethylenediaminetetraacetic acid; DEAE, diethylaminoethyl; P¡, inorganic phosphate.…”

“…Purification of DNA Polymerase ß. Initial efforts to apply our previously published procedure (Sedwick et al, 1972;Wang et al, 1974) to the purification of the hepatic enzyme were unsuccessful because polymerase ß was found to chromatograph anomalously on DEAE-cellulose. Specifically, a substantial fraction of this enzyme activity (>50%) adsorbed to the resin and eluted coincidentally with DNA polymerase a in a 0.3 KP04 (pH 8.2) step.2 This splitting of DNA polymerase ß activity was disadvantageous to its further purification, and thus we developed a new protocol that is modified from those earlier described by ourselves (Wang et al, 1974) and Chang (1973a).…”

“…Portions of fraction VI were introduced into 12 mL of a 5 to 50% glycerol gradient containing 2% BioLytes in the pH 3-10 range and 0.4% BioLytes in the pH 8-10 range. Isoelectric focusing was carried out for 5 days in a J tube as described (Wang et al, 1974), using either of two methods. To prepare enzyme for enzymological studies, method A was used, where the cathode solution was in 65% glycerol and the anode solution in 5% glycerol.…”