Role of DNA‐RNA Hybrids in Eukaryotes

A ribonuclease H activity which degrades RNA‐DNA hybrids has been partially purified from extracts of Saccharomyces cerevisiae. The activity is easily detected in the crude extracts of exponentially growing cells. The purified enzyme is a small, basic, considerably heat resistant protein which has the following properties: (a) it shows no activity with single‐stranded RNA or synthetic double‐stranded RNA but rapidly hydrolyses the RNA moiety of RNA‐DNA hybrids; (b) the action of the enzyme is predominantly and, may be exclusively, endonucleolytic in character: it will, therefore, remove hybridized RNA covalently bound to a DNA chain; (c) extensive degradation of the hybrid yields a limit digest of oligoribonucleotides which dissociate from the DNA strand and become resistant to further cleavage and (d) the enzyme is most active at alkaline pH and requires Mg 2+ and thiol groups for optimum activity. Reagents for sulfhydryl groups, ethidium bromide and some rifampicin analogues are potent inhibitors of the reaction. An RNA‐dependent DNA polymerase activity co‐purifies with ribonuclease H but the polymerase activity can be selectively heat inactivated.