DNA polymerase activity from Tetrahymena pyriformis

Abstract

The DNA polymerase of Z’etrahymena pyrifomis was first isolated from crude extracts of the cells by Pearlman and Westergaard [ 1,2] . The specific activity of this polymerase was increased if, prior to extraction, the cells were treated with either ultraviolet irradiation, electron irradiation, methotrexate plus uridine or ethidium bromide [2&4]. Using Sephadex G-200 column chromatography in 0.5 M NaCl of crude extracts of Tetrahymena, two DNA polymerase activities were observed after ultraviolet irradiation, electron irradiation or methotrexate plus uridine treatment [3] . The enzyme fraction showing increase in specific activity was found in the mitochondria [4] . As a first step in asking questions about the relation of “nuclear” to mitochondrial DNA polymerases and about the enzymes involved in DNA replication and DNA repair [2,5] we have devised a fractionation scheme to partially purify the DNA polymerase from untreated exponentially growing cells. Using this fractionation scheme, we have attempted to fractionate the activity from ethidium bromide treated cells. Our fractionation method was based on an observation made with the DNA polymerases from sea urchin embryos [6] and yeast [7] and observed independently by us. Under conditions of low salt, the DNA polymerase aggregates into high molecular weight species, and in high salt dissociates into a low molecular weight species. This characteristic of the enzyme has enabled us to purify the enzyme approximately 85 fold. Part of the increase in specific activity in ethidium bromide treated cells is detected as a new activity peak when chromatographed in low salt on Sephadex G-200.