Die Desoxyribonucleasen von Escherichia coli und die Wirkung von Mitomycin C

Abstract

The deoxyribonucleases of Escherichia coli and the effect of mitomycin C The activities of various deoxyribonucleases of Escherichia coli have been measured in crude cell extracts, ribosomal fractions and the 105 000 × g supernatants. The free deoxyribonucleases, probably exonuclease II and III, and the RNA-bound endodeoxyribonuclease I (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5) are found mainly in the ribosome-free supernatant. The distribution of the endodeoxyribonuclease between the ribosomal fraction (10 %) and the supernatant (90 %) is independent of the growth phase of the bacterial culture and of the Mg 2+ concentration of the preparation media in the range of 0.5 to 50 mM. The activities of the deoxyribonucleases, especially of the endodeoxyribonuclease I, are highest when extracts are prepared from bacteria of the early log phase. The deoxyribonuclease activities increase with increasing concentrations of Mg 2+ in the test medium over the range 0.25 to 50 mM. Furthermore, there is an increase in deoxyribonuclease activity on lowering the Mg 2+ concentration from 0.25 to 0.05 mM. This increase is probably a consequence of the release of latent bound endodeoxyribonuclease I from s-RNA, because Mg 2+ are involved in the binding of this enzyme to s-RNA. The optimal pH for endodeoxyribonuclease I in extracts is 8.0 at a Mg 2+ concentration of 0.25 mM and 6.2 at a Mg 2+ concentration of 5.0 mM. Mitomycin C added directly to cell extracts has no influence on the deoxyribonuclease activities. In extracts of mitomycin-treated E. coli the activity of a free deoxyribonuclease, which preferentially degrades native DNA at pH 8.0 and which can be inhibited by the addition of s-RNA, is increased by 100%. The characteristics correspond to endodeoxyribonuclease I. Extracts from treated cells also contain more endodeoxyribonuclease, which can be released by ribonuclease. The results indicate that, among the different deoxyribonucleases which have been isolated from E. coli until now, the activity of the endodeoxyribonuclease I is increased in extracts of mitomycin-treated E. coli. It is assumed that this enzyme is involved in the breakdown of DNA occurring in E. coli upon treatment with this antibiotic. In a following paper evidence will be presented, that this might be a consequence of the breakdown of ribosomes and s-RNA.