YEAST MITOCHONDRIAL DEOXYRIBONUCLEASE STIMULATED BY ETHIDIUM-BROMIDE .1. PURIFICATION AND PROPERTIES

A deoxyribonuclease (EtdBr DNase), which is about 25 times more active on double-stranded DNA, in which EtdBr is intercalated has been purified from yeast isolated mitochondria. This enzyme appears to be located in the mitochondrial membrane. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified fraction yielded six major bands whereas, in native conditions, it migrates as a single band. In addition to the EtdBr DNase, this fraction contains two other DNase activities, both of which are inhibited by EtdBr at the same concentration which is used to measure the EtdBr DNase activity. One is detected on double-stranded DNA at acid pH and the other on single-stranded DNA at neutral pH. The three activities cosediment in a sucrose gradient and have similar rates of heat inacti vation. The level of stimulation of the EtdBr DNase depends on the amount of intercalated EtdBr per nucleotide, the maximal activity being reached when all the intercalated sites are occupied. More detailed studies on the mechanism of this stimulation are described in the accompanying paper (Jacquemin-Sablon, H., et al. (1979) Biochemistry 18 (following paper in this issue)). The enzyme, which cuts one strand at a time in a covalently closed circular PM2 DNA molecule, is classified as an endonuclease. Possible involvement of the EtdBr DNase in the process of “petite” induction by EtdBr was investigated by measuring the enzyme level in two mutants resistant to EtdBr mutagenesis. This enzyme was present at a normal level in both strains. © 1979, American Chemical Society. All rights reserved.