Abstract
A ribonuclease which degrades RNA specifically in RNA·DNA hybrid structures has been purified 1500-fold from Escherichia coli B and 200-fold from E. coli strain D110 pol A1, endo I-, thy-). For maximal activity, the enzyme requires Mg2+ and the presence of a sulfhydryl reagent. The enzyme is capable of digesting more than 95% of the RNA in RNA·DNA hybrids to acid-soluble products. The products are oligonucleotides with 5′-phosphate and 3′-hydroxyl termini. The enzyme acts as an endonuclease and does not require free 3′ or 5′ termini for activity. However, it cannot cleave the phosphodiester bond covalently linking ribonucleotides to DNA. With circular single stranded DNA templates deoxynucleotide incorporation catalyzed by DNA polymerase II is dependent on RNA synthesis. This reaction can be stimulated by RNase H. During these studies an additional ribonuclease H activity was detected in E. coli B and purified 160-fold. Unlike RNase H, this activity is N-ethylmaleimide-resistant and sensitive to antiserum prepared against DNA polymerase I and attacks [3′-3H,5′-32P]poly(A)·poly(dT) in the 5′ → 3′ direction.
Highlights
We have found two enzymes capable of performing this role in E. coli
Two hundred grams of E. coli B were ground with 600 g of glass beads in a Waring Blendor to a thick paste in 500 ml of a solution containing 5 RI NaCl, 0.05 M Tris HCI, pH 8, 0.1 m&f EDTA, and 0.1 rnbr dithiothreitol (Buffer B)
Snake venom phosphodiesterase [26] completely digested the pentanucleotide product to AMP at pH 8.6. These results indicated the oligonucleotide product contained 3’-hydroxyl and 5’-phosphate termini and possessed the structures
Summary
24 pM [3H]poly(A) (32 cpm per pmole, 400 nucleotides in length), 24 pM poly(dT), 40 mM Tris. The acid-insoluble material was removed by centrifugation for 2 min at 4,000 rpm in an International refrigerated centrifuge and the supernatant fluid was counted in 10 ml of scintillation fluid One unit of activity is defined as the amount of enzyme producing 1 nmole of acid-soluble material in 20 min at 30”. During purification of RNase H from E. coli strain DllO, the activity on [3H]poly(I) .poly(dC)hybrids was followed. The decrease in acid-precipitable radioactivity was determined by trichloroacetic acid precipitation as above; acid-insoluble material was collected on Gelmantype E glass fiber filters, and radioactivity measured by scintillation counting
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