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Isolation and Characterization of an Endonuclease from Escherichia coli Specific for Ribonucleic Acid in Ribonucleic Acid·Deoxyribonucleic Acid Hybrid Structures

From the ¹ From the Division of Biology, Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461

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.

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