Ribonuclease H activity

Divalent metal ions are essential for ribonuclease H activity. Two Mn(II) ions have been located in the catalytic site of ribonuclease H domain of HIV-1 reverse transcriptase in close proximity to the four acidic residues Asp443, Glu478, Asp498, and Asp549 after soaking crystals in 45 mM MnCl2 (406). 

Hizi A, Hughes SH, Shaharabany M. Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. Virology 1990 175 575-580. 

Hizi A, Tal R, Hughes SH. Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse expressed in Escherichia coli. Virology 1991 180 339-346. 

A typical antisense mRNA would be 17-25 nucleotides long and would destroy the target mRNA by binding to it and invoking ribonuclease H activity. The synthetic RNAs are, however, also quickly destroyed by other ribonucle-... 

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

Schatz O, Mous J, Le Grice SFJ. HIV-1 RT-associated ribonuclease H displays both endonuclease and 3 5 exonuclease activity. EMBO J 1990 9 1171-1176. 

Chattopadhyay D, Finzel BC, Munson SH, Evans B, Sharma SK, Strakalattis NA, et al. Crystallographic analysis of an active HIV-1 ribonuclease H domain show structural features that distinguish it from the inactive form. Acta Crystallogr 1993 D49 423-427. 

Synthesis of cDNA, usually in radiolabeled form is accomplished with reverse transcriptase, the enzyme from retroviruses that synthesize a DNA-RNA hybrid from ssRNA.570 572 A short oligo (dT) primer is usually hybridized to the 3 poly (A) tail to initiate synthesis. Reverse transcriptase also has ribonuclease (RNase H) activity and will digest away the RNA. If desired, synthesis of the second strand can be carried out by a DNA polymerase to give a complete DNA duplex. Many gene sequences have been deduced from cDNA copies. 

The ribonuclease H (RNase H) family of enzymes are ubiquitous nucleases that catalyze the hydrolysis of RNA in RNA DNA hybrids (for review, see 1). In contrast to the well-studied ribonucleases A and Tl, RNase H does not employ the 2 -OH in RNA as a nucleophile but instead activates water as the nucleophile for hydrolysis in a metal-dependent reaction. The number and role(s) of divalent metal in the RNase H reaction mechanism are still unclear. 

HIV-l reverse transcriptase (RT) possesses DNA and RNA template-dependent polymerase activity, as well as an endonucleolytic ribonuclease H (RNase H) activity responsible for degradation of the RNA in an RNA/ DNA duplex. Because RT plays a critical role in the HIV life cycle, it remains a primary therapeutic target for anti-HIV drug development. ... 

A simple procedure described for the detection of encarbohydrate moiety of ribonuclease B has a-D-mannopyranosyl residues as terminal non-reducing groups, but such residues are lost as part of an oligosaccharide on action of the endo -o-l-acet-amido-2-deoxyglucanase, agarose immobilized concanavalin A was used to separate radioactive substrate (absorbed) from radioactive product (unadsorbed). The assay was used to follow the preparation of the enzyme from Streptomyces plicatus. 

Damha, M.J., Giannaris, P.A., and Marfey, P., Antisense L/D-oligodeoxynucleotide chimeras nuclease stability, base-pairing properties, and activity at directing ribonuclease H, Biochemistry, 33, 7877,1994. 

Epstein, D. A., Czarniecki, C. W., Jacobsen, H., Friedman, R. M., and Panet, A., 1981, A mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease F activity, Eur. J. Biochem. 118 9. 

S. Moore and W. H. Stein (Rockefeller, New York) contributions to the understanding of the connection between chemical structure and catalytic activity of the active centre of the ribonuclease molecule. 

Crestfield, A.M., Stein, W.H., and Moore, S. (1963) Alkylation and identification of the histidine residues at the active site of ribonuclease. J. Biol. Chem. 238, 2413-2419. 

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