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Nucleic Acids Research 2006 34(10):2906-2913; doi:10.1093/nar/gkl368
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Published online 31 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay

Nicoletta Potenza, Vincenzo Salvatore, Annalucia Migliozzi, Valentina Martone, Valentina Nobile and Aniello Russo*

Department of Life Sciences, Second University of Naples Via Vivaldi 43, 81100 Caserta, Italy

*To whom correspondence should be addressed. Tel: +39 0823 274569; Fax: +39 0823 274571; Email: aniello.russo{at}unina2.it

Received February 27, 2006. Revised March 21, 2006. Accepted April 27, 2006.

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5'-fluorescein-labeled RNA hybridized to a complementary 3'-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a kcat/Km of 330 000 M–1 s–1, a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.


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