ABSTRACT
gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.
Subject(s)
Chromatography, Affinity/methods , DNA Polymerase II/isolation & purification , Animals , Base Sequence , Catalytic Domain , DNA Polymerase II/chemistry , DNA Polymerase II/metabolism , DNA Primers , Drosophila melanogaster/embryologyABSTRACT
During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase alpha (pol.alpha) and beta (pol.beta). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.alpha and beta. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.alpha activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.beta inhibitors. With both pol.alpha and beta, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.
Subject(s)
DNA Polymerase I/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Paclitaxel/analogs & derivatives , Taxoids/pharmacology , Animals , Cell Line, Tumor , DNA Topoisomerases/drug effects , Drosophila/enzymology , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Nucleic Acid Synthesis Inhibitors/isolation & purification , Nucleic Acid Synthesis Inhibitors/pharmacology , Paclitaxel/pharmacology , Taxoids/isolation & purification , Taxus/chemistryABSTRACT
Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.
Subject(s)
DNA Polymerase II/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Glycolipids/pharmacology , Enzyme Inhibitors/chemistry , Glycolipids/chemistry , Humans , KineticsABSTRACT
The epitopes of about 100 monoclonal antibodies against human type II DNA topoisomerase were mapped along the enzyme molecules. Although they were randomly and independently established, epitope sites were unevenly distributed the toward N-terminal or C-terminal region. We suggest that the central catalytic domain is hidden inside the molecule and inaccessible to the antigen recognition sites. Using antibodies, we demonstrate the distinct localization of isoforms of Topo II in cultured cells. Some particularly useful antibodies are listed.