Role of DNA · RNA Hybrids in Eukaryotes

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Abstract
Ribonucleases H1 and H2, purified in homogeneous form from yeast cells, specifically degrade the RNA moiety of RNA · DNA hybrids. The two enzymes are physically and functionally distinct proteins. Gel electrophoresis with sodium dodecylsulfate and gel filtration revealed that RNAase H2 is a small very basic protein composed of a single polypeptide chain of molecular weight 21000. RNAase H1, also a basic protein, is made of a polypeptide chain of molecular weight 48000 which shows a marked tendency to aggregate when subjected to polyacrylamide gel electrophoresis, gel filtration or zone sedimentation. RNAase H1 and RNAase H2 require different conditions for activity. RNAase H2 digests hybridized RNA at alkaline pH and shows an absolute requirement for divalent cations, preferably Mg2+, and for ‐SH groups. The enzyme liberates a mixture of oligonucleotides with 5′‐phosphate termini and only a small amount of 5′‐mononucleotide. It is inhibited by the ‐ SH reagent N‐ethylmaleimide. RNAase H1 is active at acid pH (around pH 6), as well as at slightly alkaline pH. It is also stimulated by divalent cations but does not need to be supplemented with ‐SH groups and is not sensitive to N‐ethylmaleimide. With (rA)n ‐ (dT)n as substrate, the main degradation product is 5′‐AMP with some dinucleotide pApA, suggesting an exonucleolytic degradation process. The possible role of RNAases H in the cell is discussed.