HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Klumpp, K. Articles by Handa, B. Search for Related Content PubMed PubMed Citation Articles by Klumpp, K. Articles by Handa, B. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 9, 6181-6188, March 3, 2000

RNA and DNA Hydrolysis Are Catalyzed by the Influenza Virus Endonuclease^*

Klaus Klumpp, Linh Doan, Noel A. Roberts, and Balraj Handa

From Roche Discovery Welwyn, 40 Broadwater Road, Welwyn Garden City, Herts AL7 3AY, United Kingdom

The influenza virus polymerase complex contains a metal ion-dependent endonuclease activity, which generates short capped RNA primer molecules from capped RNA precursors. Previous studies have provided evidence for a two-metal ion mechanism of RNA cleavage, and the data are consistent with a direct interaction of a divalent metal ion with the catalytic water molecule. To refine the model of this active site, we have generated a series of DNA, RNA, and DNA-RNA chimeric molecules to study the role of the 2'-hydroxy groups on nucleic acid substrates of the endonuclease. We could observe specific cleavage of nucleic acid substrates devoid of any 2'-hydroxy groups if they contained a cap structure (m7GpppG) at the 5'-end. The capped DNA endonuclease products were functional as primers for transcription initiation by the influenza virus polymerase. The apparent cleavage rates were about 5 times lower with capped DNA substrates as compared with capped RNA substrates. Cleavage rates with DNA substrates could be increased to RNA levels by substituting the deoxyribosyl moieties immediately 5' and 3' of the cleavage site with ribosyl moieties. Similarly, cleavage rates of RNA substrates could be lowered to DNA levels by exchanging the same two ribosyl groups with deoxyribosyl groups at the cleavage site. These results demonstrate that the 2'-hydroxy groups are not essential for binding and cleavage of nucleic acids by the influenza virus endonuclease, but small differences of the nucleic acid conformation in the endonuclease active site can influence the overall rate of hydrolysis. The observed relative cleavage rates with DNA and RNA substrates argue against a direct interaction of a catalytic metal ion with a 2'-hydroxy group in the endonuclease active site.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. Hooker, R. Strong, R. Adams, B. Handa, J. H. Merrett, J. A. Martin, and K. Klumpp A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis Nucleic Acids Res., July 1, 2001; 29(13): 2691 - 2698. [Abstract] [Full Text] [PDF]

				M. B. Leahy, D. C. Pritlove, L. L. M. Poon, and G. G. Brownlee Mutagenic Analysis of the 5' Arm of the Influenza A Virus Virion RNA Promoter Defines the Sequence Requirements for Endonuclease Activity J. Virol., January 1, 2001; 75(1): 134 - 142. [Abstract] [Full Text]